Treatment of demyelinating diseases

ABSTRACT

The present invention relates generally to methods of using nalfurafine for treating and/or preventing demyelinating disease in a subject, and in particular for treating and/or preventing multiple sclerosis (MS). Also disclosed is nalfurafine for use in treating and/or preventing MS as well as pharmaceutical compositions and unit dosage forms comprising nalfurafine for use for treating and/or preventing demyelinating disease in a subject, and in particular for treating and/or preventing MS.

CLAIM OF PRIORITY

This application is a continuation of PCT Application No.PCT/IB2019/051870, filed on Mar. 7, 2019, which claims priority toAustralian Patent Application Serial No. 2018900754, filed on Mar. 8,2018, the entire contents of both applications are hereby incorporatedby reference.

1. U.S. GOVERNMENT RIGHTS

This invention was made with government support under DA018151 awardedby the National Institutes of Health. The government has certain rightsin the invention.

2. TECHNICAL FIELD

The disclosure relates generally to the use of nalfurafine (NalF) in theprevention and treatment of demyelinating diseases, in particular,multiple sclerosis.

3. BACKGROUND

The myelin sheath covers important nerve fibres in the central andperipheral nervous system of mammals, helping to facilitate transmissionof neural impulses. Diseases that affect myelin interrupt these nervetransmissions. The developing myelin sheath can be affected bycongenital metabolic disorders such as phenylketonuria, Tay-Sachsdisease, Niemann-Pick disease, Hurler's syndrome, and Krabbe's disease.Demyelination can also occur in adults as a result of injury, metabolicdisorders, immune attack, ischemia and toxic agents.

Demyelination impairs conduction of signals to the affected nerves,causing deficiency of sensation, movement, cognition and otherfunctions. Demyelination of the central nervous system is associatedwith multiple sclerosis (MS), Devic's disease, acute disseminatedencephalomyelitis, adrenoleukodystrophy, leukoencephalopathy and Leber'soptic atrophy. Demyelination of the peripheral nervous symptom givesrise to diseases such as Guillain-Barre syndrome, chronic inflammatorydemyelinating polyneuropathy, Charcot Marie Tooth (CMT) disease andprogressing inflammatory neuropathy.

Multiple sclerosis (MS) is the most well-known demyelination disease,affecting about 2.5 million people worldwide. Sufferers endure a rangeof symptoms including fatigue, vision problems, numbness, cognitiveimpairment, incontinence, poor balance and muscle weakness, ultimatelyleading to paralysis. MS can follow four major disease courses, each ofwhich can be mild, moderate or severe:

-   -   1. Relapsing-Remitting MS (RRMS)—clearly defined attacks        (flare-ups) of worsening neurological function followed by        partial or complete remission    -   2. Primary-Progressive MS (PPMS)—slowly worsening neurological        function at variable rates, with no distinct remission    -   3. Secondary-Progressing MS (SPMS)—an initial period of RRMS is        followed by a steady worsening, with or without flare-ups and        remissions    -   4. Progressive-Relapsing MS (PRMS)—steadily worsening        neurological function with clear flare-ups and partial or no        remission.

While there is no cure for MS, many FDA approved drugs such asbeta-interferon and glatiramer acetate are used to reduce relapse ratesand the formation of new lesions. Unfortunately, current treatments arenot very successful in preventing the disability associated with MS andare more successful in treating RRMS than other types. For example,current drugs are unable to stop or reverse disease progression anddisability. Clearly, alternative treatments for MS are needed.

It is therefore an object of the present invention to go at least someway towards meeting this need in the art, to provide products andmethods useful in the treatment of the disability associated with MSand/or that are able to stop and/or reverse MS disease progression anddisability and/or to at least to provide the public with a usefulchoice.

4. SUMMARY OF THE INVENTION

In one aspect the invention provides a pharmaceutical compositioncomprising nalfurafine and pharmaceutically acceptable excipients fortreating a demyelinating disease in a subject in need thereof.

In one aspect the invention provides a pharmaceutical compositioncomprising nalfurafine and at least one pharmaceutically acceptableexcipient for use for treating a demyelinating disease in a subject inneed thereof.

In another aspect the invention provides unit dosage forms comprisingabout 0.01 to about 5 mg of nalfurafine and at least onepharmaceutically acceptable carrier or excipient. In one embodiment, theunit dosage form comprises 0.05 to about 2.0 mg of nalfurafine and atleast one pharmaceutically acceptable carrier or excipient. In oneembodiment the unit dosage form comprises about 0.15 to about 0.6 mgnalfurafine and at least one pharmaceutically acceptable carrier orexcipient.

In another aspect the invention provides a method of treating ademyelinating disease in a subject in need thereof, comprisingadministering a therapeutically effective amount of nalfurafine to thesubject.

In another aspect the invention provides a method of treating ademyelinating disease in a subject comprising identifying a subject whowould benefit from a decreased level of demyelination and administeringto the subject a therapeutically effective amount of an agent thatdecreases the level of demyelination in the subject relative to thelevel of demyelination before administering the agent, wherein the agentcomprises nalfurafine.

In another aspect the invention provides a method of treating ademyelinating disease in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of anagent that decreases the level of demyelination in the subject relativeto the level of demyelination before administering the agent, whereinthe agent comprises nalfurafine.

In another aspect the invention provides a method of increasingremyelination in a subject in need thereof, comprising administering atherapeutically effective amount of nalfurafine to the subject.

In another aspect the invention provides a method of increasingremyelination in a subject comprising identifying a subject who wouldbenefit from an increased level of remyelination and administering tothe subject a therapeutically effective amount of an agent thatincreases the level of remyelination in the subject relative to thelevel of remyelination before administering the agent, wherein the agentcomprises nalfurafine.

In another aspect the invention provides a method of increasingremyelination in a subject in need thereof, comprising administering tothe subject a therapeutically effective amount of an agent thatincreases the level of remyelination in the subject relative to thelevel of remyelination before administering the agent, wherein the agentcomprises nalfurafine.

The invention also provides a use of nalfurafine in the manufacture of amedicament for treating a demyelinating disease in a subject in needthereof.

The invention also provides a use of nalfurafine in the manufacture of amedicament for increasing remyelination in a subject in need thereof.

The invention also provides nalfurafine for use for treating ademyelinating disease.

The invention also provides nalfurafine for use for increasingremyelination.

In one embodiment the disease is a demyelinating myelinoclastic disease.

In one embodiment the disease is a demyelinating leukodystrophicdisease.

In one embodiment the demyelinating disease is a central nervous systemdemyelinating disease. In one embodiment the central nervous systemdemyelinating disease is selected from the group comprising MS(including clinically isolated syndrome; CIS), optic neuritis, Devic'sdisease, inflammatory demyelinating diseases, central nervous systemneuropathies, myelopathies like Tabes dorsalis, leukoencephalopathies,leukodystrophies, or a combination thereof.

In one embodiment the demyelinating disease is MS.

In another embodiment the demyelinating disease is a peripheral nervoussystem demyelinating disease. In one embodiment the peripheral nervoussystem demyelinating disease is elected from the group comprisingGuillain-Barre syndrome and its chronic counterpart, chronicinflammatory demyelinating polyneuropathy, anti-myelin associatedglycoprotein (MAG) peripheral neuropathy, Charcot Marie Tooth (CMT)disease, copper deficiency and progressive inflammatory neuropathy.

In another aspect the invention provides a method of attenuatingdemyelination in a subject in need thereof, comprising administering atherapeutically effective amount of nalfurafine to the subject andthereby attenuating a level of demyelination in the subject relative tothe level of demyelination when nalfurafine is not administered.

The invention also provides a use of nalfurafine in the manufacture of amedicament for attenuating demyelination in a subject in need thereof.In one embodiment, the subject is a human with MS.

The invention also provides nalfurafine for use for attenuatingdemyelination in a subject in need thereof.

In another aspect the invention provides a method of treating MS in asubject in need thereof, comprising administering a therapeuticallyeffective amount of nalfurafine to the subject.

In another aspect the invention provides a method of treating MS in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of an agent that decreases a level ofdemyelination in the subject relative to the level before administeringthe agent and/or that increases a level of remyelination in the subjectin the subject relative to the level before administering the agent,wherein the agent comprises nalfurafine.

The invention also provides a use of nalfurafine in the manufacture of amedicament for treating MS in a subject in need thereof.

The invention also provides nalfurafine for use for treating MS in asubject in need thereof.

In one embodiment the subject has RRMS. In one embodiment the subjecthas PPMS. In one embodiment the subject has, or is diagnosed as having,SPMS. In one embodiment the subject has, or is diagnosed as having,PRMS. In one embodiment the subject has, or is diagnosed as having,Clinically Isolated Syndrome (CIS).

In one embodiment the treatment of MS results in one or more clinicaloutcomes when compared to subjects not treated with nalfurafine selectedfrom the group consisting of:

-   -   (a) a decrease in MS disease progression;    -   (b) a decrease in MS disease severity;    -   (c) a decrease in nerve cell demyelination;    -   (d) a decrease in frequency or severity of relapsing MS attacks;    -   (e) a decrease in MS clinical symptoms;    -   (f) the healing of damaged nerve tissue (neuro-restoration);    -   (g) an increase in remyelination of demyelinated nerves in the        central nervous system (neuro-restoration/protection);    -   (h) the protection of damaged nerve tissue from further disease        activity (neuroprotection);    -   (i) the promotion of neuronal outgrowth (neuro-regeneration) in        the central nervous system;    -   (j) a decrease in disability caused by MS;    -   (k) an improvement of nerve function; and    -   (l) an enhanced rate of remission.

In another embodiment the treatment of MS results in a reduction of oneor more clinical symptoms of MS including, but not limited to loss ofsensitivity or changes in sensation such as tingling, pins and needlesor numbness, muscle weakness or paralysis of variable severity, verypronounced reflexes, muscle spasms, or difficulty in moving;difficulties with coordination and balance (ataxia); spasticity;problems with speech or swallowing, visual problems (nystagmus, opticneuritis or double vision), fatigue, acute or chronic pain, neuropathicpain, facial pain (trigeminal neuralgia), bladder and boweldifficulties, incontinence, reduced cognitive ability, depression,anxiety and other emotional abnormalities, sexual dysfunction, Uhthoff'sphenomenon (a worsening of symptoms due to exposure to higher than usualtemperatures), and Lhermitte's sign (an electrical sensation that runsdown the back when bending the neck).

In one aspect the invention provides a method of accelerating remissionfrom MS in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of nalfurafine to thesubject.

In one aspect the invention provides a method of accelerating remissionfrom MS in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an agent thatdecreases the level of demyelination in the subject relative to thelevel of demyelination before administering the agent, wherein the agentcomprises nalfurafine.

In one aspect the invention provides a method of accelerating remissionfrom MS in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an agent thatincreases the level of remyelination in the subject relative to thelevel of remyelination before administering the agent, wherein the agentcomprises nalfurafine.

The invention also provides a use of nalfurafine in the manufacture of amedicament for accelerating remission from MS in a subject in needthereof.

The invention also provides nalfurafine for use for acceleratingremission from MS in a subject in need thereof.

In another aspect the invention provides a method of treating ademyelinating disease in a subject comprising identifying a subject whowould benefit from a decreased level of demyelination and administeringto the subject a therapeutically effective amount of an agent thatdecreases the level of demyelination relative to the level ofdemyelination before administering the agent, wherein the agentcomprises nalfurafine.

In another aspect the invention provides a method of increasingremyelination in a subject comprising identifying a subject who wouldbenefit from an increased level of remyelination and administering tothe subject a therapeutically effective amount of an agent thatincreases the level of remyelination relative to the level ofremyelination before administering the agent, wherein the agentcomprises nalfurafine.

In the above methods of the invention:

In one embodiment the therapeutically effective amount for a subject isequivalent to a dose of about 0.003 to about 0.3 mg/kg/day in mice.

In one embodiment the subject is human. In one embodiment the methodcomprises administering about 0.01 to about 5 μg nalfurafine daily,about 0.01 to about 4 μg, about 0.01 to about 3 μg, about 0.01 to about2.5 μg, about 0.01 to about 2 μg, about 0.01 to about 1.5 μg, about 0.01to about 1 μg, about 0.01 to about 0.75 μg, about 0.01 to about 0.5 μg,or about 0.25 μg nalfurafine daily.

In some embodiments the method comprises administering less than about 1μg nalfurafine, preferably less than 1 ug nalfurafine daily.

In some embodiments the method comprises a long duration therapy.

In some embodiments the long duration therapy comprises administrationof a therapeutically effective dose of nalfurafine to a subject in needthereof for at least 5 days, at least 6 days, or at least 7 days.

In some embodiments a long duration therapy comprises administration ofa therapeutically effective dose of nalfurafine to a subject in needthereof for at least a week, at least 2 weeks, at least 3 weeks, atleast 4 weeks, at least 6 weeks, or at least 8 weeks.

In some embodiments the long duration therapy comprises administrationfor at least 5 days, at least 6 days, at least 7 days, at least 14 days,at least 21 days, at least 28 days, at least 35 days, at least 42 days,at least 45 days, at least 60 days, at least 120 days, at least 240days, or at least 360 days.

In some embodiments the long duration therapy comprises a dosing gap ofat least 1 day.

Other aspects of the invention may become apparent from the followingdescription which is given by way of example only and with reference tothe accompanying figures.

In this specification where reference has been made to patentspecifications, other external documents, or other sources ofinformation, this is generally for the purpose of providing a contextfor discussing the features of the invention. Unless specifically statedotherwise, reference to such external documents is not to be construedas an admission that such documents, or such sources of information, inany jurisdiction, are prior art, or form part of the common generalknowledge in the art. However, these external documents and referencesare all cited herein by reference in their entireties or at least to theextent described herein.

It is intended that reference to a range of numbers disclosed herein(for example, 1 to 10) also incorporates reference to all rationalnumbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5,7, 8, 9 and 10) and also any range of rational numbers within that range(for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, allsub-ranges of all ranges expressly disclosed herein are hereby expresslydisclosed. These are only examples of what is specifically intended andall possible combinations of numerical values between the lowest valueand the highest value enumerated are to be considered to be expresslystated in this application in a similar manner.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition range, allintermediate ranges and subranges, as well as all individual valuesincluded in the ranges given are intended to be included in thedisclosure. In the disclosure and the claims, “and/or” meansadditionally or alternatively. Moreover, any use of a term in thesingular also encompasses plural forms.

5. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of example only and withreference to the drawings in which:

FIG. 1 is a graph showing the progression of disease in mice which haveexperimental autoimmune encephalomyelitis (EAE) over 45 days, whereinthe mice in Example 1 were treated with 0.01, 0.03, 0.1 or 0.3 mg/kgnalfurafine daily from onset (day 17).

FIGS. 2A-B are two graphs showing the total disability of EAE mice over(A) 45 days and (B) 18 days wherein the mice in Example 2 were treatedwith 0.03, 0.1 or 0.3 mg/kg nalfurafine daily from onset (day 17).

FIG. 3 is a graph showing the % weight change of EAE mice in Example 3over 45 days wherein the mice were treated with 0.03, 0.1 or 0.3 mg/kgnalfurafine daily from onset (day 17).

FIGS. 4A-C are three graphs showing immune cell infiltration into thebrain of EAE mice in Example 4 after 45 days, wherein the mice weretreated with 0.03, 0.1 or 0.3 mg/kg nalfurafine daily from onset (day17).

FIG. 5 is a graph showing the progression of disease in EAE mice inExample 5 over 45 days, wherein the mice, which had not yet developedEAE, were treated with 0.03, 0.1 or 0.3 mg/kg nalfurafine daily fromonset (day 17).

FIGS. 6A-C are a series of Transmission Electron Microscope (TEM) imagesof spinal cord sections from EAE mice in Example 6 after 45 days,wherein the mice were treated with 0.03 mg/kg nalfurafine daily fromonset (day 17).

FIG. 7 is a graph showing weight gain over 65 days of mice in Example 7treated with 0.3% cuprizone for 5 weeks, wherein the mice were treatedwith 0.1 mg/kg nalfurafine daily from week 4.

FIG. 8 is a graph showing the rotarod performance score of mice inExample 8 at 9 weeks treated with cuprizone for 5 weeks, wherein themice were treated with 0.1 mg/kg nalfurafine daily from week 4.

FIGS. 9A-D are a series of TEM imagines of the corpus callosum of micein Example 9 at 9 weeks treated with cuprizone for 5 weeks, wherein themice were treated with 0.1 mg/kg nalfurafine daily from week 4.

FIG. 10 shows that nalfurafine promotes functional recovery fromparalysis when administered therapeutically (at disease onset) in theexperimental autoimmune encephalomyelitis (EAE) model of MS.

FIG. 11 shows that nalfurafine is not effective when administeredtherapeutically as a short 4-day course starting at disease onset in EAEmodel of MS.

FIG. 12 shows that nalfurafine does not alter peak disease whenadministered therapeutically in the EAE model of MS.

FIG. 13 shows that nalfurafine promotes full recovery from EAE-inducedparalysis when administered therapeutically.

FIG. 14 shows that nalfurafine promotes full recovery from EAE-inducedparalysis when administered therapeutically with an EC50 for % recoveryof <0.001 mg/kg.

FIG. 15 shows that nalfurafine promotes sustained recovery fromEAE-induced paralysis when administered therapeutically.

FIG. 16 shows that nalfurafine also promotes functional recovery fromparalysis in male mice when administered therapeutically in EAE model ofMS.

FIG. 17 shows that nalfurafine also promotes full recovery in male micewhen administered therapeutically in EAE model of MS.

FIG. 18 shows that nalfurafine promotes sustained recovery in male micefrom EAE-induced paralysis when administered therapeutically.

FIGS. 19A-B show that nalfurafine reduces the immune cell infiltrationinto the brain when administered therapeutically in the EAE model of MS(A) whereas U 50488 does not (B).

FIG. 20 shows that myelination is improved in mice treated withnalfurafine after the onset of paralysis in the EAE model of MS.

FIGS. 21A-C show that nalfurafine does not alter the proportion of majorlymphocyte populations in the spleen during the chronic phase of EAE.

FIGS. 22A-D show that nalfurafine does not alter the overall number ofCD4 T helper cells in the spleen but shifts the CD4 T cells from aneffector to memory phenotype being suggestive of immune resolutionduring the chronic phase of EAE.

FIG. 23 shows that nalfurafine reduces disease but does not enable fullrecovery when the kappa opioid receptor (KOR) is blocked.

FIGS. 24A-C show that activation of the KOR is required for fullrecovery from paralysis mediated by nalfurafine.

FIGS. 25A-D show that myelination is improved in mice treated withnalfurafine after the onset of paralysis in the EAE model of MS.

FIGS. 26A-C show that nalfurafine treatment decreases cellularinfiltration into the spinal cord when administered therapeutically inthe EAE model of MS.

FIGS. 27A-1, 27A-2, and 27B show that nalfurafine treatment reduces thelevel of activated astrocytes in the spinal cord when administeredtherapeutically in the EAE model of MS.

FIGS. 28A-C show that nalfurafine treatment enhances recovery fromweight loss when administered therapeutically in the cuprizone model ofMS.

FIGS. 29A-G show that nalfurafine treatment enhances remyelination inthe brain when administered after demyelination in the cuprizonedemyelination disease model of MS.

FIGS. 30A-B show that nalfurafine is more effective at promotingfunctional recovery than clemastine fumarate, a known remyelinatingdrug.

FIGS. 31A-1, 31A-2, 31B-1, and 31B-2 show that nalfurafine promotes agreater and more sustained recovery than clemastine fumarate, a knownremyelinating drug.

FIGS. 32A-1, 32A-2, and 32B show that nalfurafine promotes recovery inpain threshold when administered after demyelination in the cuprizonedemyelination disease model of MS.

6. DETAILED DESCRIPTION 6.1 Nalfurafine

Nalfurafine is a drug commonly prescribed for treatment of uremicpruritus in people with chronic kidney disease. It is a non-narcoticopioid with selective K-opioid receptor (KOR) agonist activity. Theinventors have now found that nalfurafine is a surprisingly effectivetreatment for demyelinating diseases.

The generic name “nalfurafine” refers to the compound:

The IUPAC name for nalfurafine is(E)-N-[(4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-1,2,4,5,6,7,7a,13-octahydro-4,12-methanobenzofuro[3,2-e]isoquinoline-7-yl]-3-(furan-3-yl)-N-methylprop-2-enamide.Its CAS number is 152657-84-6. Nalfurafine HCl may also be referred toas17-cyclopropylmethyl-3,14-beta-dihydroxy-4,5-alpha-epoxy-6beta-(N-methyl-trans-3-(3-furyl)acrylamido)morphinanhydrochloride, TRK 820, AC-820 and MT-9938.

As used herein the term “nalfurafine” refers to the compound identifiedabove as well as to its pharmaceutically acceptable salts and solvates.

The term “pharmaceutically acceptable salts” refers to salts preparedfrom pharmaceutically acceptable non-toxic bases or acids includinginorganic or organic bases and inorganic or organic acids. Salts derivedfrom inorganic bases include aluminum, ammonium, calcium, copper,ferric, ferrous, lithium, magnesium, manganic salts, manganous,potassium, sodium, zinc, and the like. Particularly preferred are theammonium, calcium, magnesium, potassium, and sodium salts. Salts in thesolid form may exist in more than one crystal structure and may also bein the form of hydrates. Salts derived from pharmaceutically acceptableorganic non-toxic bases include salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines, and basic ion exchange resins, suchas arginine, betaine, caffeine, choline, N,N′-dibenzylethylene-diamine,diethylamine, 2-diethylaminoethanol, 2-dimethylamino-ethanol,ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, and the like. When nalfurafine is basic,salts can be prepared from pharmaceutically acceptable non-toxic acids,including inorganic and organic acids. Such acids include acetic,benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic,fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic,lactic, maleic, malic, mandelic, ethanesulfonic, mucic, nitric, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicacid, and the like. Particularly preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric, fumaric, and tartaric acids.

The term “solvate” refers to an aggregate that consists of a solute ionor molecule with one or more solvent molecules. “Solvates” includehydrates, that is, aggregates of a compound of interest with water.

Nalfurafine can be purchased from small molecule suppliers such as MedChem Express, Monmouth Junction and New Jersey, USA; AdooQ BioScience,Irvine Calif., USA.

6.2 Pharmaceutical Compositions of Nalfurafine

There is a lack of effective treatments for demyelinating diseases,including MS, and in particular, there are few effective agents that actto reduce demyelination and/or to increase remyelination. Surprisingly,the inventors have found that pharmaceutical compositions containingnalfurafine can be used to treat demyelination diseases including butnot limited to MS by acting to increase remyelination and/or to decreasedemyelination.

Accordingly, in one aspect the invention provides a pharmaceuticalcomposition comprising nalfurafine and pharmaceutically acceptableexcipients for treating a demyelinating disease in a subject in needthereof.

In another aspect the invention provides a pharmaceutical compositioncomprising nalfurafine and at least one pharmaceutically acceptableexcipient for use for treating a demyelinating disease in a subject inneed thereof.

This term “pharmaceutical composition” as used herein encompasses aproduct comprising one or more active agents, and pharmaceuticallyacceptable excipients comprising inert ingredients, as well as anyproduct which results, directly or indirectly, from combination,complexation or aggregation of any two or more of the ingredients, orfrom dissociation of one or more of the ingredients, or from other typesof reactions or interactions of one or more of the ingredients. Ingeneral, pharmaceutical compositions are prepared by bringing the activeagent into association with a liquid carrier, a finely divided solidcarrier or both, and then, if necessary, shaping the product into thedesired formulation. Said compositions are prepared according toconventional mixing, granulating, or coating methods, respectively, andcontain a percentage (%) of the active ingredient and can be determinedby a skilled worker in view of the art.

The term “comprising” as used herein means “consisting at least in partof”. When interpreting each statement in this specification thatincludes the term “comprising”, features other than that or thoseprefaced by the term may also be present. Related terms such as“comprise” and “comprises” are to be interpreted in the same manner.

The term “consisting essentially of” as used herein means the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristic(s) of the claimed invention.

The term “consisting of” as used herein means the specified materials orsteps of the claimed invention, excluding any element, step, oringredient not specified in the claim.

By “pharmaceutically acceptable excipient” or “pharmaceuticallyacceptable carrier” it is meant that the excipient or carrier must becompatible with the other ingredients of the formulation and not harmfulto the subject to whom the composition is administered.

Pharmaceutical compositions as described herein can be administeredtopically, orally or parenterally.

For example, the pharmaceutical compositions can be administered orally,including sublingually, in the form of capsules, tablets, elixirs,solutions, suspensions, or boluses formulated to dissolve in, forexample, the colon or duodenum. The formulations can comprise excipientssuch as starch or lactose or flavouring, preserving or colouring agents.

The pharmaceutical compositions can be injected parenterally, forexample, intravenously, intramuscularly or subcutaneously. Forparenteral administration, the compositions can be formulated in asterile aqueous solution or suspension that optionally comprises othersubstances, such as salt or glucose.

The compositions can be administered topically, in the form of sterilecreams, gels, pour-on or spot-on formulations, suspensions, lotions,ointments, dusting powders, drug-incorporated dressings, shampoos,collars or transdermal patches. For example, the compositions asdescribed herein can be incorporated into a cream comprising an aqueousor oily emulsion of polyethylene glycols or liquid paraffin; an ointmentcomprising a white wax soft paraffin base; a hydrogel with cellulose orpolyacrylate derivatives or other suitable viscosity modifiers; a drypowder; aerosol with butane, propane, HFA, or CFC propellants; adressing, such as, a tulle dressing, with white soft paraffin orpolyethylene glycol impregnated gauze dressings or with hydrogel,hydrocolloid, or alginate film dressings. The compositions can also beadministered intra-ocularly as an eye drop with appropriate buffers,viscosity modifiers (for example, cellulose derivatives), andpreservatives (for example, benzalkonium chloride).

The pharmaceutical compositions as described herein can also beincorporated into a transdermal patch comprising nalfurafine. Details ofsuch patches can be found in, for example, WO2015/025766, WO2015/025767,WO2016/208729, WO2017/094337 and WO2017/170933, the details of which areincorporated by reference herein.

For oral administration, capsules, boluses, or tablets can be preparedby mixing the pharmaceutical compositions as described herein with asuitable finely divided diluent or carrier, additionally containing adisintegrating agent and/or binder such as starch, lactose, talc, ormagnesium stearate.

For parenteral administration injectable formulations can be prepared inthe form of a sterile solution or emulsion.

The compositions described herein can be presented in unit dosage formand can be prepared by any of the methods well known in the art ofpharmacy. The term “unit dosage form” means a single dose wherein allactive and inactive ingredients are combined in a suitable system, suchthat the patient or person administering the drug can open a singlecontainer or package with the entire dose contained therein and does nothave to mix any components together from two or more containers orpackages. Typical examples of unit dosage forms are tablets or capsulesfor oral administration or transdermal patches comprising the unitdosage. These examples of unit dosage forms are not intended to belimiting in any way, but merely to represent typical examples in thepharmacy arts of unit dosage forms.

In another aspect the invention provides unit dosage forms comprisingabout 0.01 to about 5 mg of nalfurafine and at least onepharmaceutically acceptable carrier or excipient. In one embodiment, theunit dosage form comprises 0.05 to about 2.0 mg of nalfurafine and atleast one pharmaceutically acceptable carrier or excipient. In oneembodiment the unit dosage form comprises about 0.15 to about 0.6 mgnalfurafine and at least one pharmaceutically acceptable carrier orexcipient.

In one aspect the invention provides a unit dosage form comprising about0.1 to about 10 μg of nalfurafine and at least one pharmaceuticallyacceptable carrier or excipient. In one embodiment the unit dosage formcomprises about 0.5 to about 7.5 μg nalfurafine, about 0.75 to about 5μg nalfurafine, about 1 to 4 μg nalfurafine, about 2-3 μg nalfurafine,about 2 μg nalfurafine, about 3 μg nalfurafine, about 4 μg nalfurafineor about 5 μg nalfurafine.

In one embodiment the unit dosage form comprises less than about 2 μg,1.5 μg, 1.0 μg, 0.5 μg, 0.25 μg or 0.1 μg, preferably less than 2 μg,1.5 μg, 1.0 μg, 0.5 μg, 0.25 μg or 0.1 μg.

In another embodiment, the unit dosage form is for treating ademyelinating disease in a subject in need thereof, preferably whereinthe subject has MS. In another embodiment, the unit dosage is formulatedfor treating a demyelinating disease in a subject in need thereof. Inone embodiment the demyelinating disease is MS.

In another embodiment the unit dose is formulated for increasingremyelination in a subject in need thereof, preferably wherein thesubject has MS.

In one embodiment, the unit dosage form is for oral administration,preferably the unit dosage form is formulated for oral administration.In another embodiment, the unit dosage form is a transdermal patch.

The term “about” as used herein means a reasonable amount of deviationof the modified term such that the end result is not significantlychanged. For example, when applied to a value, the term should beconstrued as including a deviation of +/−5% of the value.

Pharmaceutical compositions of nalfurafine can be used in combinationwith other therapies for treating demyelination diseases.

6.3 Therapeutic Uses of Nalfurafine

The inventors have surprisingly found that nalfurafine gives rise tomany positive effects in demyelination in MS mouse models. For example,the inventors have found that nalfurafine is effective at treatingdemyelination in mouse models of EAE and cuprizone-induceddemyelination, results that are translatable to treating demyelinatingdiseases such as MS in humans. The inventors have also found thatnalfurafine is unexpectedly effective at increasing remyelination insubjects in need thereof. Accordingly, this drug, which has a provensafety record, could be highly beneficial in the treatment ofdemyelination diseases and/or for increasing remyelination.

As set out in Examples 1, 10 and 12-18, nalfurafine promotes functional(including full and sustained) recovery from EAE-induced paralysis inmale and female mice. Nalfurafine also reduces EAE-induced totaldisability (see Example 2) and promotes recovery from EAE-induced weightloss (see Example 3). Importantly, the disease score is reducedcompletely in the examples described herein to <0.5, which is consideredto represent a “full recovery” from paralysis in the art, with oneexception. A short 4-day time course starting at disease outset was noteffective at promoting recovery (Example 11), demonstrating the efficacyof a long duration therapy as described herein.

Nalfurafine reduces immune cell infiltration into the brain in the EAEmodel of MS (see Example 4) and is more effective than the comparatorU-50488, which does not (Example 19. When administered before onset,nalfurafine promotes functional recovery from paralysis, in the EAEmodel of MS (see Example 5). Myelination is also improved in micetreated with nalfurafine after the onset of paralysis in the EAE modelof MS (Examples 6, 20 and 25).

By the examples described herein the inventors show clearly thatnalfurafine induces and/or increases remyelination in the EAE model. InExample 6, TEM images of the spinal cords of EAE mice treated withnalfurafine resemble those of the healthy control.

The EAE results were confirmed by cuprizone studies described inExamples 7-9 and 11. In Examples 7 and 28, nalfurafine improved weightgain when administered after cuprizone-induced demyelination. In Example8, nalfurafine enhanced the functional recovery of coordination andbalance in demyelinated mice. Remyelination of the corpus callosumoccurred when cuprizone-treated mice were administered nalfurafine (seeExamples 9 and 29).

In Example 15 the demonstration of sustained recovery is noteworthy andshows the quite unexpected ability of nalfurafine to reverse, in asustained manner, the symptoms of demyelination. This surprising resultindicates that nalfurafine can mediate sustained recovery ofdemyelinating diseases including MS.

In Example 21, nalfurafine does not deplete the major immune cellpopulations in the periphery despite reducing immune cell infiltrationinto the brain. In example 22, nalfurafine promotes a switch in T helpercells from effector to memory cells suggestive of immune responseresolution.

In Examples 23 and 24, the KOR is required for the full effect ofnalfurafine but nalfurafine is effective at reducing diseaseindependently of the KOR suggesting the full mechanism by whichnalfurafine exerts its effects is more complex than KOR activation.

The positive effects of nalfurafine on mice were particularly surprisingat dosages of 0.003 mg/kg to 0.3 mg/kg, which can be converted to anequivalent human dose using the Regan-Shaw equation (Reagan-Shaw S;Nihal M; Ahmad N: Dose translation from animal to human studiesrevisited, FASEB J. 2007, Oct. 17).

Alternatively, dosages of 0.003 to 0.3 mg/kg can be converted to anequivalent human dose using the method of interspecies comparisondescribed herein.

The skilled worker in the art appreciates that there are alternativealgorithms that can be used to convert an observed therapeutic dosagefrom a mouse model into an equivalent human dose once the effectivemouse dosage has been demonstrated. Such algorithms can be usedeffectively by the skilled person to determine the appropriate humandose

For example, using a method of interspecies comparison, a skilled workeremploys the ratio of the efficacy dose for itch vs the efficacy dose forMS in the same species. This ratio can be applied to the human dose toconvert dosage for itch to the dosage for MS. In this case, there isdose data for treating itch in both mouse and human models, and thisenables the calculations described below.

61/968,897 Data describing the drug dose that produces 50% of themaximal effect (ED₅₀).

Complete Route of ED₅₀ Inhibition Mouse Model Administration (μg/kg)(μg/kg) Reference Substance P IV 3.77 7.5-10  Winfuran-Assessmentinduced report European Medicines scratch Agency, Committee forSubstance P SC 1.65 10 Medicinal Products for induced Human Use scratch(EMA/CHMP/138212/2014) Substance P PO 9.61 100 induced (66%) scratchMorphine SC 2.34  5-10 induced scratch Histamine PO 7.3  30-100 Togashiet al. (2002). induced Antipruritic activity of the scratch κ-opioidreceptor agonist, Substance P PO 19.6 100 TRK-820. Eur 3 Pharmacolinduced 435:259 scratch

For itch model the average in vivo efficacy ED₅₀ is ˜2.71 μg/kg (roundedup to 3 μg/kg) by SC or IV administration (only the data in the top tworows of the table above were used in this calculation). The rationalefor this is:

-   -   Administration in our EAE study was intraperitoneal (i.p.)    -   Bioavailability of nalfurafine (as described in        Winfuran—Assessment report European Medicines Agency, Committee        for Medicinal Products for Human Use (EMA/CHMP/138212/2014):        -   oral (PO) administration is ˜32%        -   subcutaneous (s.c.) is 96%        -   intravenous (IV) is 100%    -   Therefore, s.c. and IV administration will have a similar        bioavailability to i.p., whereas PO administration will not due        to first-pass effect of hepatic metabolism, and therefore it has        been excluded from the calculations. Additionally, the morphine        induced scratch model works through a mechanism of action        unrelated to that of substance P itch, and therefore was        excluded.        Converting Dosage for Itch vs EAE

The calculation assumes that itch response is a biomarker (surrogate)for EAE.

-   -   1. Mouse dose for itch is 3 μg/kg/day    -   2. Mouse dose for EAE is 3 μg/kg/day (effective dose shown in        FIG. 10)    -   3. Therefore, the ratio of itch to EAE in mouse=1    -   4. Using the ratio of efficacy for itch vs EAE in the same        species (mouse) of 1    -   5. The effective dose in humans for itch of 2.5 μg/body/day    -   6. Calculation to convert EAE mouse to Human dose prediction:        EAE mouse dose/(3 μg/kg/day mouse itch×2.5 μg/body/day human        itch)=Human MS dose.    -   7. Conversion of EAE mouse dose to predicted human MS dose:        -   1. 3 μg/kg/day mouse=2.5 μg/body/day for human (FIG. 10)        -   2. 10 μg/kg/day mouse=8.33 μg/body/day for human (FIG. 1)        -   3. 30 μg/kg/day mouse=25 μg/body/day for human (FIG. 1)        -   4. 100 μg/kg/day mouse=83.33 μg/body/day for human (FIG. 1)        -   5. 300 μg/kg/day mouse=250 μg/body/day for human (FIG. 1)

As many demyelinating diseases cause horribly debilitating symptoms, anyimprovement in treatment outcomes provides an important development. Theinventors have discovered that nalfurafine is an effective treatment fordemyelinating diseases, and in particular MS. In one example, theinventors believe that treatment with nalfurafine will be effective foralleviating the debilitating symptoms related to Clinically IsolatedSyndrome (CIS). One of the MS disease courses, CIS generally refers to afirst episode of neurologic symptoms associated with MS. Typically, thisinitial episode is caused by inflammation or demyelination in thecentral nervous system (CNS), and will last 24 hours or more.

Therefore, in one aspect, the invention provides a method of treating ademyelinating disease in a subject in need thereof, comprisingadministering a therapeutically effective amount of nalfurafine to thesubject.

In another aspect the invention provides a method of treating ademyelinating disease in a subject comprising identifying a subject whowould benefit from a decreased level of demyelination and administeringto the subject a therapeutically effective amount of an agent thatdecreases the level of demyelination in the subject relative to thelevel of demyelination before administering the agent, wherein the agentcomprises nalfurafine.

In another aspect the invention provides a method of treating ademyelinating disease in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of anagent that decreases the level of demyelination in the subject relativeto the level of demyelination before administering the agent, whereinthe agent comprises nalfurafine.

The term “treating” as used herein with reference to a disease orcondition refers to the following: (a) ameliorating the disease orcondition such as by eliminating or causing regression of or decreasingthe severity of the disease or medical condition of the subject beingtreated relative to an untreated subject according to art-acceptedcriteria for monitoring the disease or condition (Wattjes et al. (2015).Evidence-based guidelines: MAGNIMS consensus guidelines on the use ofMRI in multiple sclerosis—establishing disease prognosis and monitoringpatients. Nat. Rev. Neurol. 11, 597-606; Traboulsee et al. (2016).Revised Recommendations of the Consortium of MS Centers Task Force for aStandardized MRI Protocol and Clinical Guidelines for the Diagnosis andFollow-Up of Multiple Sclerosis. AJNR Am. 3. Neuroradiol. 37, 394-401;Toosy et al. (2014). Optic neuritis. Lancet Neurol. 13, 83-99; Ontanedaet al. (2017). Clinical outcome measures for progressive MS trials.Mult. Scler. 23, 1627-1635; Naismith et al. (2012). Diffusion tensorimaging in acute optic neuropathies: predictor of clinical outcomes.Arch. Neurol. 69, 65-71); (b) suppressing the disease or condition suchas by slowing or arresting the development of the disease or conditionrelative to an untreated subject according to art-accepted criteria formonitoring the disease or condition (Oh et al. (2019). Imaging outcomemeasures of neuroprotection and repair in MS: A consensus statement fromNAIMS. Neurology; Sormani et al. (2017). Assessing Repair in MultipleSclerosis: Outcomes for Phase II Clinical Trials. Neurother. 3. Am. Soc.Exp. Neurother. 14, 924-933; Zhang et al. (2018). Clinical trials inmultiple sclerosis: milestones. Ther. Adv. Neurol. Disord. 11; Bjartmaret al. (2003). Axonal loss in the pathology of MS: consequences forunderstanding the progressive phase of the disease. 3. Neurol. Sci. 206,165-171; Toosy et al. (2014). Optic neuritis. Lancet Neurol. 13, 83-99)or (c) alleviating a symptom of the disease or condition in the subjectrelative to an untreated subject according to art-accepted criteria formonitoring the disease or condition (van Munster et al. (2017). OutcomeMeasures in Clinical Trials for Multiple Sclerosis. CNS Drugs 31,217-236; Uitdehaag (2018). Disability Outcome Measures in Phase IIIClinical Trials in Multiple Sclerosis. CNS Drugs 32, 543-558; Toosy etal. (2014). Optic neuritis. Lancet Neurol. 13, 83-99). In some preferredembodiments “treating” refers to ameliorating as in (a), suppressing asin (b) and/or alleviating as in (c) in a statistically significantmanner relative to an appropriate untreated control subject according toart-accepted criteria for monitoring the disease or condition.

In the definition of “treating” the art accepted criteria are one ormore of Criteria for measuring disability may include the expandeddisability scale, multiple sclerosis functional composite Z-score andmultiple sclerosis Impact Scale and Medical Outcomes Study Short Form,imaging of the brain, spinal cord or optic nerve, Multiple SclerosisFunctional Composite, and novel composite measures of disability, inaddition to tests evaluating manual dexterity, ambulation, vision(including measures of axial diffusivity, visual acuity, contrastsensitivity, visual evoked potentials (VEPs), and thickness of theretinal nerve fiber layer (RNFL) and cognition.

The subject may show an observable or measurable decrease in one or moreof the symptoms associated with or related to the disease or conditionas known to those skilled in the art, as indicating improvement. In someembodiments, the disease or condition is a demyelinating disease,preferably MS, and the subject shows an observable and measurabledecrease in one or more of the symptoms associated with or related toMS, preferably a decrease in demyelination as known to those skilled inthe art, as indicating improvement. In preferred embodiments theimprovement is a statistically significant improvement relative to anappropriate untreated control subject according to art-accepted criteriafor monitoring the disease or condition.

The terms “decrease” and “reduced” (and grammatical variations thereof)as used herein with reference to demyelination mean any measurable orobservable reduction in an amount or level of demyelination or of anysymptom of a demyelinating disease that is attributable to demyelinationin a treated subject relative to the level of demyelination in anappropriate control (e.g., untreated) subject. In preferred embodimentsthe measurable or detectable decrease or reduction is a statisticallysignificant decrease or reduction, relative to an appropriate control.

The term “increase” (and grammatical variations thereof as used hereinwith reference to demyelination means any measurable or observableincrease in an amount or level of remyelination or an improvement of anysymptom of a demyelinating disease that is attributable to remyelinationin a treated subject relative to the level of remyelination in anappropriate control (e.g., untreated) subject; e.g., placebo ornon-active agent. An example of quantifying remyelination isdemonstrated with treatment with clemastine fumarate using measures ofVEPs to evaluate remyelination and recovery. (Green et al. (2017)Clemastine fumarate as a remyelinating therapy for multiple sclerosis(ReBUILD): a randomised, controlled, double-blind, crossover trial.Lancet. 390, 2481-2489; Jankowska-Lech et al. (2019). Peripapillaryretinal nerve fiber layer thickness measured by optical coherencetomography in different clinical subtypes of multiple sclerosis. Mult.Scler. Relat. Disord. 27, 260-268; Naismith et al. (2012). Diffusiontensor imaging in acute optic neuropathies: predictor of clinicaloutcomes. Arch. Neurol. 69, 65-71; Oh et al. (2019). Imaging outcomemeasures of neuroprotection and repair in MS: A consensus statement fromNAIMS. Neurology; Sormani et al. (2017). Assessing Repair in MultipleSclerosis: Outcomes for Phase II Clinical Trials. Neurother. 3. Am. Soc.Exp. Neurother. 14, 924-933. In preferred embodiments the measurable ordetectable reduction is a statistically significant reduction, relativeto an appropriate control.

The terms “administration of” or “administering” should be understood tomean providing nalfurafine or a pharmaceutical composition comprising,consisting essentially of, or consisting of, nalfurafine to the subjectin need of treatment in a therapeutically useful form for the mode ofadministration. Nalfurafine can be administered via any suitable route.Potential routes of administration include without limitation oral,parenteral (including intramuscular, subcutaneous, intradermal,intravenous, intraarterial, intramedullary and intrathecal),intraperitoneal, and topical (including dermal/epicutaneous,transdermal, mucosal, transmucosal, intranasal (e.g., by nasal spray ordrop), intraocular (e.g., by eye drop), pulmonary (e.g., by inhalation),buccal, sublingual, rectal and vaginal.

The term “therapeutically” as used herein means “at disease onset”.

In certain embodiments, nalfurafine is administered via oral dosageforms such as tablets, capsules, syrups, suspensions, and the like. Inanother embodiment, nalfurafine is administered via a transdermal patch.

The term “therapeutically effective amount” refers to a sufficientquantity of the active agent, in a suitable composition, and in asuitable dosage form to treat the noted disease conditions or to obtaina measurable or observable result such as a decrease in demyelination oran increase in remyelination. The “therapeutically effective amount”will vary depending on the compound, the severity of the demyelinationdisease, and the species, age, weight, etc., of the subject to betreated.

In one embodiment, the therapeutically effective amount of nalfurafineis the amount equivalent to about 0.003-about 0.3 mg/kg in a mouse whichcan be converted according to accepted practice into an animal or humansubject dosage. For example, using the Reagan-Shaw equation, atherapeutically effective amount of nalfurafine for a dog would be about0.67-about 2 mg/kg.

In one embodiment, the therapeutically effective amount of nalfurafineis the amount equivalent to about 0.003-about 0.3 mg/kg in a mouse,converted according the method of interspecies comparison describedherein. In one embodiment a therapeutically effective amount ofnalfurafine for a human is about 0.01 to about 5 μg nalfurafine daily,preferably about 0.01 to about 2.5 μg nalfurafine daily.

In one embodiment the subject is human. In one embodiment the methodcomprises administering about 0.01 to about 5 μg nalfurafine daily,about 0.01 to about 4 μg, about 0.01 to about 3 μg, about 0.01 to about2.5 μg, about 0.01 to about 2 μg, about 0.01 to about 1.5 μg, about 0.01to about 1 μg, about 0.01 to about 0.75 μg, about 0.01 to about 0.5 μg,or about 0.25 μg nalfurafine daily.

In one embodiment the method comprises administering about 0.01 to about2.5 μg nalfurafine daily, about 0.025 to about 2 μg, about 0.05 to about1 μg, about 0.075 to about 0.75 μg, about 0.1 to about 0.5 μg, or about0.225 to about 0.325 μg nalfurafine daily.

In some embodiments the method comprises administering less than about 1μg nalfurafine daily, preferably less than 1 ug nalfurafine daily.

In one embodiment the method comprises administering about 0.01 to about0.1 μg nalfurafine daily, about 0.025 to about 0.075 μg, about 0.06 toabout 0.04 μg, or about 0.05 μg nalfurafine daily.

In one embodiment the method comprises a long duration therapy.

In some embodiments the long duration therapy comprises administrationof a therapeutically effective dose of nalfurafine to a subject in needthereof for at least 5 days, at least 6 days, or at least 7 days.

In some embodiments the long duration therapy comprises administrationof a therapeutically effective dose of nalfurafine to a subject in needthereof for at least 5, preferably at least 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, preferably at least 90 days.

In some embodiments a long duration therapy comprises administration ofa therapeutically effective dose of nalfurafine to a subject in needthereof for at least a week, at least 2 weeks, at least 3 weeks, atleast 4 weeks, at least 6 weeks, or at least 8 weeks.

In some embodiments the long duration therapy comprises administrationfor at least 5 days, at least 6 days, at least 7 days, at least 14 days,for at least 21 days, for at least 28 days, for at least 35 days, for atleast 42 days, for at least 45 days, for at least 60 days, for at least120 days, for at least 240 days, or for at least 360 days.

In some embodiments a long duration therapy comprises administration ofa therapeutically effective dose of nalfurafine to a subject in needthereof for at least 1 week, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or at least 52 weeks.

In some embodiments a long duration therapy comprises administration ofa therapeutically effective dose of nalfurafine to a subject in needthereof for at least 1 month, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, or at least 36 months.

In some embodiments the long duration therapy comprises a dosing gap,preferably wherein the dosing gap is at least 1 day.

In some embodiments dosing gap comprises at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, or 14 days.

In some embodiments the dosing gap comprises at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 weeks.

In some embodiments the dosing gap comprises at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 months.

The term “demyelinating disease” refers to a disease of the nervoussystem in which the myelin sheath of neurons is damaged. Demyelinatingdiseases include demyelinating myelinoclastic diseases and demyelinatingleukodystrophic diseases. Treatment of a demyelinating disease cancomprise treatment with an agent that decreases demyelination and/or anagent that increases remyelination.

Demyelinating diseases may affect the central nervous system andperipheral nervous system. The central nervous system demyelinatingdiseases include multiple sclerosis including clinically isolatedsyndrome (CIS) optic neuritis, Devic's disease, inflammatorydemyelinating diseases, central nervous system neuropathies like thoseproduced by Vitamin B12 deficiency, myelopathies like Tabes dorsalis,leukoencephalopathies like progressive multifocal leukoencephalopathy,leukodystrophies, or a combination thereof. The peripheral nervoussystem demyelinating diseases include Guillain-Barre syndrome and itschronic counterpart, chronic inflammatory demyelinating polyneuropathy,anti-MAG peripheral neuropathy, Charcot Marie Tooth (CMT) disease,copper deficiency, progressive inflammatory neuropathy, or a combinationthereof. The term “subject” refers to a mammal, more preferably a human,or companion animal. Preferred companion animals include cats, dogs andhorses. Other mammalian subjects include agricultural animals, includinghorses, pigs, sheep, goats, cows, deer, or fowl: and laboratory animal,including monkeys, rats, mice, rabbits and guinea pig.

The invention also provides a use of nalfurafine in the manufacture of amedicament for treating a demyelinating disease in a subject in needthereof.

The invention also provides a use of nalfurafine in the manufacture of amedicament for increasing remyelination in a subject in need thereof.

The invention also provides nalfurafine for use for treating ademyelinating disease.

The invention also provides nalfurafine for use for increasingremyelination.

In one embodiment the disease is a demyelinating myelinoclastic disease.

In one embodiment the disease is a demyelinating leukodystrophicdisease.

In one embodiment the demyelinating disease is a central nervous systemdemyelinating disease. In one embodiment the central nervous systemdemyelinating disease is selected from the group comprising MS(including clinically isolated syndrome; CIS), optic neuritis, Devic'sdisease, inflammatory demyelinating diseases, central nervous systemneuropathies, myelopathies like Tabes dorsalis, leukoencephalopathies,leukodystrophies, or a combination thereof.

In one embodiment the demyelinating disease is MS.

In another embodiment the demyelinating disease is a peripheral nervoussystem demyelinating disease. In one embodiment the peripheral nervoussystem demyelinating disease is elected from the group comprisingGuillain-Barre syndrome and its chronic counterpart, chronicinflammatory demyelinating polyneuropathy, anti-myelin associatedglycoprotein (MAG) peripheral neuropathy, Charcot Marie Tooth (CMT)disease, copper deficiency and progressive inflammatory neuropathy.

In another aspect the invention provides a method of increasingremyelination in a subject in need thereof, comprising administering atherapeutically effective amount of nalfurafine to the subject.

In another aspect the invention provides a method of increasingremyelination in a subject comprising identifying a subject who wouldbenefit from an increased level of remyelination and administering tothe subject a therapeutically effective amount of an agent thatincreases the level of remyelination in the subject relative to thelevel of remyelination before administering the agent, wherein the agentcomprises nalfurafine.

In another aspect the invention provides a method of increasingremyelination in a subject in need thereof, comprising administering tothe subject a therapeutically effective amount of an agent thatincreases the level of remyelination in the subject relative to thelevel of remyelination before administering the agent, wherein the agentcomprises nalfurafine.

Specifically contemplated as embodiments of the invention describedherein relating to a method of increasing remyelination in a subject areall of the embodiments of the invention set forth herein relating to theaspects of the invention that are methods of decreasing demyelination,methods of treating MS, methods of attenuating demyelination, methods ofaccelerating remission of MS, and methods of treating a demyelinatingdisease.

In another aspect the invention provides a method of attenuatingdemyelination in a subject in need thereof, comprising administering atherapeutically effective amount of nalfurafine to the subject andthereby attenuating a level of demyelination in the subject relative tothe level of demyelination when nalfurafine is not administered.

In another aspect the invention provides a method of attenuatingdemyelination in a subject in need thereof, comprising administering atherapeutically effective amount of an agent that decreases the level ofdemyelination in the subject relative to the level of demyelinationbefore administering the agent and/or that increases the level ofremyelination in the subject relative to the level of remyelinationbefore administering the agent wherein the agent comprises nalfurafine.

In one embodiment the subject is human. In one embodiment the methodcomprises administering about 0.01 to about 5 μg nalfurafine daily,about 0.01 to about 4 μg, about 0.01 to about 3 μg, about 0.01 to about2.5 μg, about 0.01 to about 2 μg, about 0.01 to about 1.5 μg, about 0.01to about 1 μg, about 0.01 to about 0.75 μg, about 0.01 to about 0.5 μg,or about 0.25 μg nalfurafine daily.

In one embodiment the method comprises administering about 0.01 to about2.5 μg nalfurafine daily, about 0.025 to about 2 μg, about 0.05 to about1 μg, about 0.075 to about 0.75 μg, about 0.1 to about 0.5 μg, or about0.225 to about 0.325 μg nalfurafine daily.

In some embodiments the method comprises administering less than about 1μg nalfurafine daily, preferably less than 1 ug nalfurafine daily.

In one embodiment the method comprises administering about 0.01 to about0.1 μg nalfurafine daily, about 0.025 to about 0.075 μg, about 0.06 toabout 0.04 μg, or about 0.05 μg nalfurafine daily.

The term “attenuation of demyelination” means in certain embodimentsthat the amount or level of demyelination in the subject as a result ofthe disease or as a symptom of the disease is reduced when compared tootherwise identical conditions in an appropriate control subject or atan appropriate control reference timepoint and/or in certain embodimentsthat the amount or level of remyelination in the subject is increasedwhen compared to an otherwise identical conditions in an appropriatecontrol subject or at an appropriate control reference timepoint. Insome preferred embodiments the reduction or increase as compared to theappropriate control is a statistically significant reduction orincrease.

In certain preferred embodiments, the term “attenuation ofdemyelination” thus means that the amount of or level demyelination inthe subject as a result of the disease or as a symptom of the disease isreduced or decreased in a statistically significant manner when comparedto a suitable control as would be understood by a person of skill in theart in view of the present disclosure and/or the amount or level ofremyelination in the subject is increased in a statistically significantmanner when compared to a suitable control as would be understood by aperson of skill in the art in view of the present disclosure.

Similarly, the term “improvement in nerve function” refers to aquantifiable improvement in function having a statistically differentchange in a measurable parameter relative to an appropriate control asrecognized by a person of skill in the art. In some embodiments theimprovement in function has a statistically significant change in themeasurable parameter. In one embodiment the measurable parameter is thedisease score as described in Example 1.

Symptoms attributable to demyelination will vary depending on thedisease but may include, for example but not limited to, neurologicaldeficits, such as chronic pain, cognitive impairment (including memory,attention, conceptualization and problem-solving skills) and informationprocessing; paresthesia in one or more extremities, in the trunk, or onone side of the face; weakness or clumsiness of a leg or hand; or visualdisturbances, e.g. partial blindness and pain in one eye (retrobulbaroptic neuritis), dimness of vision, or scotomas.

The invention also provides a use of nalfurafine in the manufacture of amedicament for attenuating demyelination in a subject in need thereof.

The invention also provides nalfurafine for use for attenuatingdemyelination in a subject in need thereof.

In another aspect the invention provides a method of treating MS in asubject in need thereof, comprising administering a therapeuticallyeffective amount of nalfurafine to the subject. The subject can sufferfrom any type of MS including CIS, RRMS, PRMS, SPMS, PRMS or MS thatfollows a different and/or undefined disease course.

The invention also provides a use of nalfurafine in the manufacture of amedicament for treating MS in a subject in need thereof.

The invention also provides nalfurafine for use for treating MS in asubject in need thereof.

In one embodiment the subject has RRMS. In one embodiment the subjecthas PPMS. In one embodiment the subject has, or is diagnosed as having,SPMS. In one embodiment the subject has, or is diagnosed as having,PRMS. In one embodiment the subject has, or is diagnosed as having,Clinically Isolated Syndrome (CIS).

In another aspect the invention provides a method of treating MS in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of an agent that decreases a level ofdemyelination in the subject relative to the level before administeringthe agent and/or that increases a level of remyelination in the subjectin the subject relative to the level before administering the agent,wherein the agent comprises nalfurafine.

In some embodiments the methods of treating MS set forth herein cancomprise one or more of the following steps selected from the groupconsisting of diagnosing MS in the subject, testing for demyelination inthe subject, testing for a reduction or reversal in demyelination in thesubject, testing for remyelination in the subject, testing for a levelof paralysis or a reduction or reversal of a level of paralysis in thesubject, and testing for a decrease or increase of coordination and/orbalance in the subject.

In one embodiment a method of treating MS and/or of treating ademyelinating disease and/or of attenuating demyelination and/or ofincreasing remyelination comprises identifying a subject who wouldbenefit from a level of decreased demyelination.

In some embodiments a subject who would benefit from a level ofdecreased demyelination and/or a level of increased remyelination isidentified on the basis of exhibiting one or more clinical symptoms ofMS including, but not limited to: loss of sensitivity or changes insensation such as tingling, pins and needles or numbness, muscleweakness of variable severity, very pronounced reflexes, muscle spasms,or difficulty in moving; difficulties with coordination and balance(ataxia); spasticity; problems with speech or swallowing, visualproblems (nystagmus, optic neuritis or double vision), fatigue, acute orchronic pain, facial pain (trigeminal neuralgia), bladder and boweldifficulties, incontinence, reduced cognitive ability, depression,anxiety and other emotional abnormalities, sexual dysfunction, Uhthoff'sphenomenon (a worsening of symptoms due to exposure to higher than usualtemperatures), and Lhermitte's sign (an electrical sensation that runsdown the back when bending the neck).

In some embodiments the therapeutically effective amount of nalfurafineto be administered to a human subject is about 0.01 to about 5 mgnalfurafine daily, about 0.01 to about 4 μg, about 0.01 to about 3 μg,about 0.01 to about 2.5 μg, about 0.01 to about 2 μg, about 0.01 toabout 1.5 μg, about 0.01 to about 1 μg, about 0.01 to about 0.75 μg,about 0.01 to about 0.5 μg, or about 0.25 μg nalfurafine daily.

In some embodiments the therapeutically effective amount of nalfurafineto be administered to a human subject is about 0.01 to about 2.5 μgnalfurafine daily, about 0.025 to about 2 μg, about 0.05 to about 1 μg,about 0.075 to about 0.75 μg, about 0.1 to about 0.5 μg, or about 0.225to about 0.325 μg nalfurafine daily.

In some embodiments the method comprises administering less than about 1μg nalfurafine daily, preferably less than 1 ug nalfurafine daily.

In some embodiments the therapeutically effective amount of nalfurafineto be administered to a human subject is about 0.01 to about 0.1 μgnalfurafine daily, about 0.025 to about 0.075 μg, about 0.06 to about0.04 μg, or about 0.05 μg nalfurafine daily.

In one embodiment the treatment results in one or more clinical outcomesas compared to subjects not treated with nalfurafine, selected from thegroup consisting of:

-   -   (a) a decrease in MS disease progression;    -   (b) a decrease in MS disease severity;    -   (c) a decrease in nerve cell demyelination;    -   (d) a decrease in frequency or severity of relapsing MS attacks;    -   (e) a decrease in MS clinical symptoms;    -   (f) the healing of damaged nerve tissue (neuro-restoration);    -   (g) an increase in remyelination of demyelinated nerves in the        central nervous system (neuro-restoration/protection);    -   (h) the protection of damaged nerve tissue from further disease        activity (neuroprotection);    -   (i) the promotion neuronal outgrowth (neuro-regeneration) in the        central nervous system;    -   (j) a decrease in disability caused by MS;    -   (k) an improvement of nerve function; and    -   (l) an enhanced rate of remission.

In another embodiment the treatment results in a reduction of one ormore clinical symptoms of MS including, but not limited to loss ofsensitivity or changes in sensation such as tingling, pins and needlesor numbness, muscle weakness of variable severity, very pronouncedreflexes, muscle spasms, or difficulty in moving; difficulties withcoordination and balance (ataxia); spasticity; problems with speech orswallowing, visual problems (nystagmus, optic neuritis or doublevision), fatigue, acute or chronic pain, facial pain (trigeminalneuralgia), bladder and bowel difficulties, incontinence, reducedcognitive ability, depression, anxiety and other emotionalabnormalities, sexual dysfunction, Uhthoff's phenomenon (a worsening ofsymptoms due to exposure to higher than usual temperatures), andLhermitte's sign (an electrical sensation that runs down the back whenbending the neck).

In one aspect the invention provides a method of accelerating remissionof MS in a subject in need thereof, the method comprising administeringa therapeutically effective amount of nalfurafine to the subject.

In one aspect the invention provides a method of accelerating remissionfrom MS in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an agent thatdecreases the level of demyelination in the subject relative to thelevel of demyelination before administering the agent, wherein the agentcomprises nalfurafine.

In one aspect the invention provides a method of accelerating remissionfrom MS in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an agent thatincreases the level of remyelination in the subject relative to thelevel of remyelination before administering the agent, wherein the agentcomprises nalfurafine.

The invention also provides a use of nalfurafine in the manufacture of amedicament for accelerating remission from MS in a subject in needthereof.

The invention also provides nalfurafine for use in acceleratingremission from MS in a subject in need thereof.

In some embodiments the therapeutically effective amount of nalfurafineto be administered to a human subject is about 0.01 to about 5 μgnalfurafine daily, about 0.01 to about 4 μg, about 0.01 to about 3 μg,about 0.01 to about 2.5 μg, about 0.01 to about 2 μg, about 0.01 toabout 1.5 μg, about 0.01 to about 1 μg, about 0.01 to about 0.75 μg,about 0.01 to about 0.5 μg, or about 0.25 μg nalfurafine daily.

In some embodiments the therapeutically effective amount of nalfurafineto be administered to a human subject is about 0.01 to about 2.5 μgnalfurafine daily, about 0.025 to about 2 μg, about 0.05 to about 1 μg,about 0.075 to about 0.75 μg, about 0.1 to about 0.5 μg, or about 0.225to about 0.325 μg nalfurafine daily.

In some embodiments the method comprises administering less than about 1μg nalfurafine daily, preferably less than 1 ug nalfurafine daily.

In some embodiments the therapeutically effective amount of nalfurafineto be administered to a human subject is about 0.01 to about 0.1 μgnalfurafine daily, about 0.025 to about 0.075 μg, about 0.06 to about0.04 μg, or about 0.05 μg nalfurafine daily.

The term “enhanced remission of MS” as used herein, means that the startof the remission process is reached faster and/or the rate at whichremission is achieved is faster (as compared to subjects not treatedwith nalfurafine).

Remission of MS can be measured using any technique known in the artincluding but not limited to physical disability status, biologicalmarkers and brain scans using MRI.

In one aspect the invention provides a method of treating MS in a humansubject in need thereof, the method comprising administering to thesubject about 0.01 to about 5 mg nalfurafine daily, about 0.05 to about2.0 mg, about 0.15 to 0.6 mg nalfurafine daily, wherein the treatmentresults in one or more clinical outcomes as compared to subjects nottreated with nalfurafine selected from the group consisting of:

-   -   (a) a decrease in MS disease progression;    -   (b) a decrease in MS disease severity;    -   (c) a decrease in nerve cell demyelination;    -   (d) a decrease in frequency or severity of relapsing MS attacks;    -   (e) a decrease in MS clinical symptoms;    -   (f) the healing of damaged nerve tissue (neuro-restoration);    -   (g) an increase in remyelination of demyelinated nerves in the        central nervous system (neuro-restoration/protection);    -   (h) the protection of damaged nerve tissue from further disease        activity (neuroprotection);    -   (i) the promotion neuronal outgrowth (neuro-regeneration) in the        central nervous system;    -   (j) a decrease in disability caused by MS;    -   (k) an improvement of nerve function; and    -   (l) an enhanced rate of remission.

In one aspect the invention provides a method of treating MS in a humansubject in need thereof, the method comprising administering to thesubject about 0.01 to about 5 μg nalfurafine daily, about 0.01 to about4 μg, about 0.01 to about 3 μg, about 0.01 to about 2.5 μg, about 0.01to about 2 μg, about 0.01 to about 1.5 μg, about 0.01 to about 1 μg,about 0.01 to about 0.75 μg, about 0.01 to about 0.5 μg, or about 0.25μg nalfurafine daily, wherein the treatment results in one or moreclinical outcomes as compared to subjects not treated with nalfurafineselected from the group consisting of:

-   -   (a) a decrease in MS disease progression;    -   (b) a decrease in MS disease severity;    -   (c) a decrease in nerve cell demyelination;    -   (d) a decrease in frequency or severity of relapsing MS attacks;    -   (e) a decrease in MS clinical symptoms;    -   (f) the healing of damaged nerve tissue (neuro-restoration);    -   (g) an increase in remyelination of demyelinated nerves in the        central nervous system (neuro-restoration/protection);    -   (h) the protection of damaged nerve tissue from further disease        activity (neuroprotection);    -   (i) the promotion neuronal outgrowth (neuro-regeneration) in the        central nervous system;    -   (j) a decrease in disability caused by MS;    -   (k) an improvement of nerve function; and    -   (l) an enhanced rate of remission.

In some embodiments the therapeutically effective amount of nalfurafineto be administered to a human subject is about 0.01 to about 2.5 μgnalfurafine daily, about 0.025 to about 2 μg, about 0.05 to about 1 μg,about 0.075 to about 0.75 μg, about 0.1 to about 0.5 μg, or about 0.225to about 0.325 μg nalfurafine daily.

In some embodiments the method comprises administering less than about 1μg nalfurafine daily, preferably less than 1 ug nalfurafine daily.

In some embodiments the therapeutically effective amount of nalfurafineto be administered to a human subject is about 0.01 to about 0.1 μgnalfurafine daily, about 0.025 to about 0.075 μg, about 0.06 to about0.04 μg, or about 0.05 μg nalfurafine daily.

In another aspect the invention provides a method of treating ademyelinating disease in a subject comprising identifying a subject whowould benefit from a decreased level of demyelination and administeringto the subject a therapeutically effective amount of an agent thatdecreases the level of demyelination relative to the level ofdemyelination before administering the agent, wherein the agentcomprises nalfurafine.

In another aspect the invention provides a method of increasingremyelination in a subject comprising identifying a subject who wouldbenefit from an increased level of remyelination and administering tothe subject a therapeutically effective amount of an agent thatincreases the level of remyelination relative to the level ofremyelination before administering the agent, wherein the agentcomprises nalfurafine.

Specifically contemplated as embodiments of the invention describedherein relating to nalfurafine for use in decreasing demyelination,attenuating demyelination, accelerating remission of MS, treating MS,treating a demyelinating disease and increasing remyelination are all ofthe embodiments of the invention set forth herein relating to theaspects of the invention that are methods of decreasing demyelination,attenuating demyelination, accelerating remission of MS, treating MS,treating a demyelinating disease and increasing remyelination.

Additionally, specifically contemplated as embodiments of the inventiondescribed herein relating to the use of nalfurafine in the manufactureof a medicament for decreasing demyelination, attenuating demyelination,accelerating remission of MS, treating MS or for increasingremyelination are all of the embodiments of the invention set forthherein relating to the aspects of the invention that are methods ofdecreasing demyelination, attenuating demyelination, acceleratingremission of MS, treating MS, treating a demyelinating disease andincreasing remyelination.

In addition, specifically contemplated herein for all recited method,use and nalfurafine for use aspects of the invention are all of theembodiments set out herein that relate to long duration therapy anddosing gaps in long duration therapy.

The invention consists in the foregoing and also envisages constructionsof which the following gives examples only and in no way limit the scopethereof.

6.4 Examples Example 1: Nalfurafine Promotes Functional Recovery fromParalysis when Administered Therapeutically in the ExperimentalAutoimmune Encephalomyelitis (EAE) Model of MS

Experimental detail: Female, C57BL/6 mice were immunized subcutaneously(s.c.) in the hind flanks to induce EAE using myelin oligodendrocyteglycoprotein (MOG) peptide 35-55 (50 mg/mouse) in complete Freund'sadjuvant containing heat-killed Mycobacterium tuberculosis (500μg/mouse). In addition, pertussis toxin (200 ng/mouse) was administeredintraperitoneally (i.p.) on days 0 and 2. Mice were weighed and scoreddaily. On day 17 (vertical dotted line in FIG. 1), mice were started ondaily treatment with vehicle only (Veh; 10% tween and 10% DMSO insaline) or nalfurafine at 0.3, 0.1, 0.03, or 0.01 mg/kg by i.p.injection. Nalfurafine was obtained from the University of Kansas,Synthetic Chemical Biology Core Laboratory (97.6% pure by HPLC).Treatment allocation was blinded. The disease was scored from 0-5 with 0(normal), 1 (partial tail paralysis), 2 (full tail paralysis), 3 (onehind limb paralysed or severe disability in both hind limbs), 4(complete paralysis of both hind limbs) and 5 (moribund). This model isa standard disease model for multiple sclerosis and is described inWhite et al. 2018. Scientific Reports. 8:259 which is incorporatedherein by reference in its entirety. Shown in FIG. 1 are resultscombined from 2 independent experiments. **** p<0.0001 &* p<0.05 byone-way ANOVA with Dunnett's multiple comparison test.

Interpretation and impact: The results demonstrate that nalfurafine isable to treat on-going disease. The reduction of disease in allnalfurafine-treated groups indicates recovery from paralysis, which iscomplete at some doses (0.1 and 0.03 mg/kg) and unusual in this model.Finally, the dose at which nalfurafine shows the most rapid recovery inthis example is 0.1 mg/kg with doses above and below this levelappearing less effective.

Example 2: Nalfurafine Reduces Total Disability when AdministeredTherapeutically in the EAE Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. On day 17, mice were started on daily treatment withvehicle only (Veh) or nalfurafine at 0.3, 0.1, or 0.03 mg/kg by i.p.injection. The area under the curve (AUC) was calculated for each mousebased upon the daily disease score and represents the total disabilityexperienced. Shown in FIGS. 2 A-B are results from 1 representativeexperiment. * p<0.05 by one-way ANOVA with Dunnett's multiple comparisontest.

Interpretation and impact: Despite all treatment groups having similardisease scores at the start of treatment (lower graph), mice treateddaily with nalfurafine had significantly lower total disability by day45 after immunization to induce EAE (upper graph). Doses of 0.03 and 0.1mg/kg nalfurafine had the greatest effect at reducing disability. The0.1 mg/kg nalfurafine dose results in a 60% reduction in disease.

Without wishing to be bound by theory, the inventors believe that theresults in Example 2 highlight the benefits of treatment withnalfurafine over a period of at least a week. Accordingly, in someembodiment's administration comprises administration for at least 7days, at least 14 days, at least 30 days, at least 45 days, at least 60days, at least 120 days, at least 240 days, or at least 360 days.

Example 3: Nalfurafine Promotes Recovery from EAE-Induced Weight Losswhen Administered Therapeutically

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Mice were weighed daily and the % change in body weightcalculated. On day 17 (vertical dotted line in FIG. 3), mice werestarted on daily treatment with vehicle only (Veh) or nalfurafine at0.3, 0.1, or 0.03 mg/kg by i.p. injection.

Interpretation and impact: As shown in FIG. 3, at onset of disease, micerapidly lose weight. Once treatment with nalfurafine is initiated(vertical dotted line), mice recover from EAE-induced weight loss.

Example 4: Nalfurafine Reduces the Immune Cell Infiltration into theBrain when Administered at Low Doses Therapeutically in the EAE Model ofMS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. On day 17, mice were started on daily treatment withvehicle only (Veh) or nalfurafine at 0.3, 0.1, or 0.03 mg/kg by i.p.injection. On day 45 after immunization to induce EAE, mice were culled,and immune cells isolated from the brains. Isolation was by Percollgradient as described in White et al. 2018. Scientific Reports. 8:259.Once isolated, cells were stained with fluorescently labelled antibodiesto identify specific immune cell types and analysed by flow cytometry.All infiltrating immune cells were identified by CD45^(high) expression;CD4 T cells were identified as CD45^(high)CD4+, and macrophages asCD45^(high)CD11b⁺Gr-1⁻. The relative number of cells is expressed as aratio to microglia (MG), a brain resident immune cell identified asCD45^(medium)CD11b⁺. * p<0.05 by one-way ANOVA with Dunnett's multiplecomparison test.

Interpretation and impact: As shown in FIGS. 4A-C, at day 45, there wasa significant elevation in immune cells in the brains of vehicle-treatedEAE mice compared to healthy animals. Treatment with 0.03 mg/kgnalfurafine significantly reduced the number of infiltrating immunecells suggesting that at this dose, nalfurafine can haveimmunomodulatory properties. Interestingly, while mice treated with 0.1nalfurafine had similar levels of infiltrating cells as vehicle-treatedanimals, these mice had no overt signs of disease and had recoveredfully from paralysis (FIG. 1).

Example 5: Nalfurafine Promotes Functional Recovery from Paralysis whenAdministered Before the Onset of Paralysis in the EAE Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. On day 17 (vertical dotted line in FIG. 5), mice werestarted on daily treatment with vehicle only (Veh) or nalfurafine at0.3, 0.1, or 0.03 mg/kg by i.p. injection. Shown in FIG. 5 are resultsin mice that were not sick at the time of treatment but developeddisease later. * p<0.05 by two-way ANOVA with Holm-Sidak's multiplecomparison test.

Interpretation and impact: Treating with nalfurafine prior to diseaseonset did not alter the onset of disease. However, treatment withnalfurafine led to a rapid recovery from paralysis compared tovehicle-treated mice. These data suggest that treating with nalfurafinewill also be effective at reducing total disability if administeredbefore disease but may not prevent onset.

Example 6: Myelination is Improved in Mice Treated with Nalfurafineafter the Onset of Paralysis in the EAE Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. On day 17, mice were started on daily treatment withvehicle only or nalfurafine at 0.03 mg/kg by i.p. injection. On day 45after immunization to induce EAE, mice were culled, and spinal cordswere processed for transmission electron microscopy (TEM). Shown inFIGS. 6A-C are representative TEM images of spinal cord sections from ahealthy (A), vehicle-treated EAE (B), or nalfurafine-treated EAE mouse(C) stained to show that dark myelin rings around the nerve axons.

Interpretation and impact: At day 45, there was a significant reductionin the dark stained myelin in the spinal cord of the vehicle-treated EAEmice suggesting demyelination has occurred. Additionally, the nerveaxons appear bloated and the cytoplasm disorganized suggesting cellularstress. In contrast, the nerve axons appear healthy and well-myelinatedin the nalfurafine-treated mouse, which is concordant with fullfunctional recovery.

Example 7: Nalfurafine Improved Weight Gain when Administered afterDemyelination in the Cuprizone Model of Demyelination

Experimental detail: Female, C57BL/6 mice were fed 0.3% cuprizone in thediet for 5 weeks to induce demyelination. At the start of week 4(vertical dashed line in FIG. 7), mice were started on daily treatmentwith vehicle only or nalfurafine 0.1 mg/kg by i.p. injection. At thestart of week 5 (vertical dotted line in FIG. 7), cuprizone was removedfrom the diet to enable spontaneous remyelination. Mice were weigheddaily and the % weight change calculated.

Interpretation and impact: As shown in FIG. 7, cuprizone causedsignificant weight loss in mice as previously reported. This weight losswas reversed significantly more effectively by administration ofnalfurafine than vehicle alone.

Example 8: Nalfurafine Enhances the Functional Recovery of Coordinationand Balance when Administered after Demyelination in the Cuprizone Modelof Demyelination

Experimental detail: Female, C57BL/6 mice were fed 0.3% cuprizone in thediet for 5 weeks to induce demyelination and treated with nalfurafine asdescribed in Example 7. Behavioural tests including the rotarod assay,which measures coordination, were performed weekly. Mice were trained onan accelerating rotarod apparatus (Panlab, Harvard Apparatus) over aperiod of 4 to 5 days before recording baseline latencies at day 0followed by weekly measurements throughout cuprizone treatment andrecovery. The rotarod was set to 4 rotations per minute (rpm) and anacceleration rate of 40 rpm with a maximum cut-off time of 5 minutes.The time and speed at which the animal falls off the rotating rod wasrecorded and the average of 3 replicates recorded. Data showsperformance at week 9 following Veh or nalfurafine (0.1 mg/kg) treatmentrelative to performance at week 5. * p<0.05 by Students t-test.

Interpretation and impact: As shown in FIG. 8, cuprizone impairedcoordination in mice as previously reported. Cuprizone-induceddisability was reversed by administration of nalfurafine. These datasuggest that nalfurafine is effective at reducing disability in a modelof non-immune mediated demyelination such as that found in someprogressive MS patients.

Example 9: Nalfurafine Enhances Myelination when Administered afterDemyelination in the Cuprizone Model of Demyelination

Experimental detail: Female, C57BL/6 mice were fed 0.3% cuprizone in thediet for 5 weeks to induce demyelination as described in Example 7. Onday 65, mice were culled, and brains were processed for transmissionelectron microscopy (TEM). Shown are representative TEM images ofsections from the corpus callosum of a healthy (no cuprizone),vehicle-treated & cuprizone-treated, or nalfurafine-treated &cuprizone-treated mouse stained to show the dark myelin rings around thenerve axons. Myelin was quantified by g-ratio, which is the inner axonaldiameter divided by the total outer diameter.

Interpretation and impact: As shown in FIGS. 9 A-D, cuprizone caused aloss of myelin and a concurrent disruption in regular axonal structuresin the corpus callosum compared to healthy controls. In contrast, moremyelin was detected, and the structure was less disorganized in thecorpus callosum of animals treated with cuprizone & nalfurafine. Thesedata indicate that nalfurafine treatment promotes remyelination andrepair after cuprizone-induced, non-immune-mediated demyelination. Asimilar non-immune associated demyelination occurs in some progressiveMS patients.

Example 10: Nalfurafine Promotes Functional Recovery from Paralysis whenAdministered Therapeutically in the Experimental AutoimmuneEncephalomyelitis (EAE) Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIG. 10. On the day of disease onset(score ≥1, dotted line), mice were started on daily treatment withvehicle only (Veh) or nalfurafine at 0.3, 0.1, 0.03, 0.01, or 0.003mg/kg by i.p. injection. Treatment allocation was blinded. Shown are thealigned scores from mice (n=33 in Veh, 3 in 0.3, 4 in 0.1, 5 in 0.03, 20in 0.01, and 4 in 0.003 mg/kg groups) starting from onset/treatmentinitiation. One animal in the 0.3 mg/kg nalfurafine group and 2 from thevehicle group were euthanized at day 17-18. ****p<0.0001 by two-wayANOVA all doses (except 0.3 mg/kg) compared to vehicle.

Interpretation and impact: By treating after the onset of disease(paralysis), we show that nalfurafine is able to treat on-going disease.The reduction of disease in all nalfurafine-treated groups indicatesrecovery from paralysis, which is complete at some doses (0.01 and 0.03mg/kg); full recovery from disease is unusual in this model and theefficacy of the nalfurafine treatment is surprising. Finally, the doseat which nalfurafine shows the most rapid recovery in this example is0.01 mg/kg, and this finding has been replicated in 6 independentexperiments.

Example 11: Nalfurafine is not Effective when Administered as a Short4-Day Course Starting at Disease Onset in EAE Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIG. 11. On the day of disease onset(score ≥1, dotted line), mice were started on daily treatment withvehicle only or nalfurafine at 0.01 mg/kg by i.p. injection dailythroughout the experimental course or only for four days (shaded area).Shown are the aligned scores from mice (n=5/group) starting fromonset/treatment initiation. **p<0.01 by two-way ANOVA NalF (fulltreatment) compared to nalfurafine (4 days) or vehicle.

Interpretation and impact: Treatment with nalfurafine does not enhancerecovery when administered for only four days starting from diseaseonset, whereas treatment with a longer duration does enhance recoveryeffectively.

Example 12: Nalfurafine does not Alter Peak Disease when AdministeredTherapeutically in the EAE Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIG. 12. On the day of disease onset(score ≥1), mice were started on daily treatment with vehicle only ornalfurafine at 0.3, 0.1, 0.03, 0.01, or 0.003 mg/kg by i.p. injection.The peak disease score during the first EAE episode was recorded andshown are the mean and standard error of individual mice (n=33 in Veh, 3in 0.3, 4 in 0.1, 5 in 0.03, 20 in 0.01, and 4 in 0.003 mg/kg groups).No significant differences were found between any nalfurafine dose andvehicle by Kruskal-Wallis with Dunn's multiple comparison test. Thesedata are from the same experiments as Example 10.

Interpretation and impact: Because no difference in peak disease scorewas found at any dose of nalfurafine compared to vehicle, nalfurafinedid not appear to alter the initial immune-mediated neuroinflammatoryevent that leads to demyelination and paralysis. This finding suggeststhat the functional improvement observed (i.e. the recovery fromparalysis) occurs because the initial insult has been repaired andperhaps not because the initial insult itself was stopped.

Example 13: Nalfurafine Promotes Full Recovery from EAE-InducedParalysis when Administered Therapeutically

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIG. 13. On the day of disease onset(score ≥1), mice were started on daily treatment with vehicle only ornalfurafine at 0.3, 0.1, 0.03, 0.01, or 0.003 mg/kg by i.p. injection.Mice were considered recovered if they received a score ≤0.5 by day 23post treatment initiation. Shown are the percentages of mice in eachgroup that recovered (n=33 in Veh, 3 in 0.3, 4 in 0.1, 5 in 0.03, 20 in0.01, and 4 in 0.003 mg/kg groups). ****p<0.0001, **p<0.01, and *p<0.05by Fisher's exact test. These data are from the same experiments asExample 10.

Interpretation and impact: Treatment with nalfurafine enables fullfunctional recovery (i.e. no paralysis) when administeredtherapeutically and at a wide range of doses (0.003-0.1 mg/kg all show asignificant effect). Full recovery in this model of disease is unusual.The efficacy achieved with the treatment of nalfurafine isextraordinary.

Example 14: Nalfurafine Promotes Full Recovery from EAE-InducedParalysis when Administered Therapeutically with an EC₅₀ of <0.001 Ma/Kg

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIG. 14. On the day of disease onset(score ≥1), mice were started on daily treatment with vehicle only ornalfurafine at 0.1, 0.03, 0.01, or 0.003 mg/kg by i.p. injection. Micewere considered recovered if they received a score ≤0.5 by day 23 posttreatment initiation. Shown are the percentages of mice in each groupthat recovered (n=33 in Veh, 4 in 0.1, 5 in 0.03, 20 in 0.01, and 4 in0.003 mg/kg groups). A dose-response curve has been fitted from a doseof 0.1 mg/kg, in which 100% recovered, to the vehicle alone, in which12.1% recovered. This curve calculates an EC₅₀ of <0.001 mg/kg. Thesedata are from the same experiments as Example 13.

Interpretation and impact: Treatment with Nalfurafine enables fullfunctional recovery (i.e. no paralysis) when administeredtherapeutically and at a wide range of doses (0.003-0.1 mg/kg all show asignificant effect). Full recovery in this model of disease is unusual.The efficacy achieved with the treatment of nalfurafine isextraordinary. To achieve 50% of this effect (i.e. EC₅₀) an estimateddose of <0.001 mg/kg is required.

Example 15: Nalfurafine Promotes Sustained Functional Recovery fromEAE-Induced Paralysis when Administered Therapeutically

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1, Results are shown in FIG. 15. On the day of disease onset(score ≥1), mice were started on daily treatment with vehicle only ornalfurafine at 0.3, 0.1, 0.03, 0.01, or 0.003 mg/kg by i.p. injection.Mice were considered recovered if they received a score ≤0.5 by day 23post treatment initiation. Shown are the number of days mice were inrecovery in each group (n=33 in Veh, 3 in 0.3, 4 in 0.1, 5 in 0.03, 20in 0.01, and 4 in 0.003 mg/kg groups). ****p<0.0001, **p<0.01, and*p<0.05 by one-way ANOVA with Holm-Sidak's multiple comparison test.These data are from the same experiments as Example 10.

Interpretation and impact: Treatment with nalfurafine enables asustained functional recovery (i.e. no paralysis) when administeredtherapeutically and at a wide range of doses (0.003-0.1 mg/kg all show asignificant effect).

Example 16: Nalfurafine Promotes Functional Recovery from Paralysis inMale Mice when Administered Therapeutically in EAE Model of MS

Experimental detail: EAE was induced in male C57BL/6 mice as describedin Example 1. Results are shown in FIG. 16. On the day of disease onset(score ≥1, line), mice were started on daily treatment with vehicle onlyor nalfurafine at 0.01 mg/kg by i.p. injection. Treatment allocation wasblinded. Shown are the aligned scores from mice (n=5/group) startingfrom onset/treatment initiation. ****p<0.0001 by two-way ANOVA comparedto vehicle.

Interpretation and impact: Nalfurafine is effective at enablingfunctional recovery from paralysis in both females and males.

Example 17: Nalfurafine Promotes Full Recovery in Male Mice whenAdministered Therapeutically in EAE Model of MS

Experimental detail: EAE was induced in male C57BL/6 mice as describedin Example 1. Results are shown in FIG. 17. On the day of disease onset(score ≥1), mice were started on daily treatment with vehicle only ornalfurafine at 0.01 mg/kg by i.p. injection. Mice were consideredrecovered if they received a score ≤0.5 by day 23 post treatmentinitiation. Shown are the percentages of mice in each group thatrecovered (n=5/group). **p<0.01 by Fisher's exact test. These data arefrom the same experiments as Example 16.

Interpretation and impact: Treatment with nalfurafine promotes fullrecovery (i.e. no paralysis) in both female and male when administeredtherapeutically.

Example 18: Nalfurafine Promotes Sustained Recovery in Male Mice fromEAE-Induced Paralysis when Administered Therapeutically

Experimental detail: EAE was induced in male C57BL/6 mice as describedin Example 1. Results are shown in FIG. 18. On the day of disease onset(score ≥1), mice were started on daily treatment with vehicle only ornalfurafine at 0.01 mg/kg by i.p. injection. Mice were consideredrecovered if they received a score ≤0.5 by day 23 post treatmentinitiation. Shown are the number of days mice were in recovery in eachgroup (n=5/group). ****p<0.0001 by Student's t test. These data are fromthe same experiments as Example 16.

Interpretation and impact: Treatment with nalfurafine enables asustained functional recovery (i.e. no paralysis) in both females andmales when administered therapeutically.

Example 19: Nalfurafine Treatment Reduces the Immune Cell Infiltrationinto the Brain when Administered Therapeutically in the EAE Model of MS(A) Whereas U-50488 does not (B)

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIGS. 19A and 19B. On the day ofdisease onset (score ≥1), mice were started on daily treatment withvehicle only or nalfurafine at 0.3, 0.1, 0.03, 0.01, or 0.003 mg/kg byi.p. injection (A). In a separate experiment, mice were similarlytreated with vehicle alone or U-50488, a KOR agonist at 1.6 and 5 mg/kg(B). During the chronic phase (>24 days post treatment initiation), micewere culled, and immune cells isolated from the brains. Isolation was byPercoll gradient as described in White et al. 2018. Scientific Reports.8:259. Once isolated, cells were stained with fluorescently labelledantibodies to identify specific immune cell types and analysed by flowcytometry. All infiltrating immune cells were identified by CD45^(high)expression. The relative number of cells is expressed as a ratio tomicroglia (MG), a brain resident immune cell identified asCD45^(medium)CD11b⁺. *p<0.05 by one-way ANOVA with Holm-Sidak's multiplecomparison test compared to vehicle. NS, not-significant.

Interpretation and impact: In the chronic stage of EAE, there was asignificant elevation in immune cells in the brains of vehicle-treatedEAE mice compared to healthy animals (A). Treatment with 0.03 and 0.01mg/kg nalfurafine significantly reduced the number of infiltratingimmune cells suggesting that at these doses, nalfurafine can haveimmunomodulatory properties. Interestingly, while mice treated with 0.1and 0.003 nalfurafine had similar levels of infiltrating cells asvehicle-treated animals, these mice had no overt signs of disease andhad recovered fully from paralysis (FIG. 13). Additionally, nalfurafinebut not U-50488 reduced neuroinflammation in this model indicating thatnot all KOR agonists have this activity (B).

Example 20: Myelination is Improved in Mice Treated with Nalfurafineafter the Onset of Paralysis in the EAE Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIG. 20. On the day of disease onset(score ≥1), mice were started on daily treatment with vehicle only ornalfurafine at 0.03 or 0.01 mg/kg by i.p. injection. During the chronicphase (>24 days post treatment initiation), mice were culled, perfusedwith 4% paraformaldehyde and spinal cords were processed for histology.Sections were stained with luxol fast blue to assess the % area of thespinal cord that is demyelinated (i.e. does not stain with luxol fastblue). % demyelination was assessed using ImageJ. Shown are the meansand standard error of individual values from vehicle (n=7) or 0.01 (n=6)and 0.03 (n=4) nalfurafine-treated EAE mice. **p<0.01 by one-way ANOVAwith Holm-Sidak's multiple comparison test.

Interpretation and impact: During the chronic phase, when nalfurafineenabled full functional recovery in mice, there was a significantreduction in the percentage of demyelination in the spinal cord of thenalfurafine-treated EAE mice suggesting remyelination may have occurred.

Example 21: Nalfurafine does not Alter the Proportion of MajorLymphocyte Populations in the Spleen During the Chronic Phase of EAE

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIGS. 21A-C. On the day of diseaseonset (score ≥1), mice were started on daily treatment with vehicle onlyor nalfurafine at 0.01 mg/kg by i.p. injection. During the chronic phase(27 days post treatment initiation), mice were culled and theirsplenocytes assessed by flow cytometry. The percentage of the majorlymphocyte populations were identified using CD4 (CD4 T helper cells),CD8 (CD8 cytotoxic T cells), and B220 (B cells), and expressed as % liveleukocytes (i.e. CD45+ cells). Shown are the means and standard error ofindividual mice with n=3 (healthy), 4 (vehicle) and 8. No significantdifferences were found between vehicle and healthy or nalfurafine byone-way ANOVA with Holm-Sidak's multiple comparison test.

Interpretation and impact: Nalfurafine do not alter the proportion ofthe major lymphocyte populations in the spleen despite reducing thenumber of infiltrating immune cells into the central nervous system. Themaintenance of normal lymphocyte numbers in the spleen in thenalfurafine treated mice indicates that nalfurafine does not reduceimmune cell infiltration into the brain by killing immune cells.

Example 22: Nalfurafine does not Alter the Overall Number of CD4 THelper Cells in the Spleen but Shifts the CD4 T Cells from an Effectorto Memory State being Suggestive of Immune Resolution During the ChronicPhase of EAE

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIGS. 22A-D. On the day of diseaseonset (score ≥1), mice were started on daily treatment with vehicle onlyor nalfurafine at 0.01 mg/kg by i.p. injection. During the chronic phase(27 days post treatment initiation), mice were culled and theirsplenocytes assessed by flow cytometry. Naïve CD4 T cells(CD4⁺CD44⁻CD62L^(high)), effector CD4 T cells (CD4⁺CD44⁺CD62L⁻), andcentral memory CD4 T cells (CD4⁺CD44⁺CD62L^(high)) are expressed as %CD4 T cells. “Teff:cm ratio” is the ratio of effector to central memoryT cells. Shown are the means and standard error of individual mice withn=3 (healthy), 4 (vehicle) and 8. **p<0.01 and *p<0.05 by one-way ANOVAwith Holm-Sidak's multiple comparison test.

Interpretation and impact: The increased effector to central memoryratio in the vehicle-treated mice with EAE compared to healthy miceindicates an on-going and active immune response mediated by CD4 Tcells. The overall number of CD4 T cells was the same betweennalfurafine and vehicle treated mice. The reduced ratio in thenalfurafine-treated compared to the vehicle-treated mice indicates ashift toward a memory phenotype which occurs during the resolution phaseof the immune response. The shift to a memory state indicates thatimmune resolution is occurring in nalfurafine-treated mice in a model ofMS where disease is driven by an active immune response.

Example 23: Nalfurafine Reduces Disease but does not Enable FullRecovery when the Kappa Opioid Receptor (KOR) is Blocked

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIG. 23. On the day of disease onset(score >1, dotted line), mice were treated with vehicle only (daily),nalfurafine (0.01 mg/kg by i.p. injection daily), the KOR antagonistnorBNI (10 mg/kg by i.p. injection weekly), or both nalfurafine andnorBNI. Shown are the aligned scores from mice (n=8-9/group) startingfrom onset/treatment initiation. ****p<0.0001 by two-way ANOVA NalFcompared to vehicle or NalF+noBNI.

Interpretation and impact: Administration of the KOR antagonist, norBNI,abolishes the ability of nalfurafine to enable full recovery fromparalysis (i.e. score <0.5), and this finding indicates that the KOR isrequired for the full effect of nalfurafine. The finding thatnalfurafine is effective at reducing disease independently of the KOR(i.e. in the presence of norBNI) indicates that the full mechanism bywhich nalfurafine exerts its effects is more complex than KORactivation.

Example 24: Activation of the KOR is Required for Full Recovery fromParalysis Mediated by Nalfurafine

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIGS. 24A-C. On the day of diseaseonset (score >1, dotted line), mice were treated with vehicle only(daily), nalfurafine (0.01 mg/kg by i.p. injection daily), the KORantagonist norBNI (10 mg/kg by i.p. injection weekly), or bothnalfurafine and norBNI. The peak disease score during the first EAEepisode was recorded, and mice were considered recovered if theyreceived a score <0.5 by day 23 post treatment initiation. Shown are thepeak disease scores, the percentage of mice in each group thatrecovered, and the number of days in recovery (n=8-9/group). **p<0.01and ****p<0.0001 by Fisher's exact test (% recovered) or one-way ANOVAwith Holm-Sidak's multiple comparison test (#days in recovery). Thesedata are from the same experiments as Example 23.

Interpretation and impact: Administration of the KOR antagonist, norBNI,abolishes the ability of nalfurafine to enable and sustain recovery fromparalysis (i.e. score <0.5), and this finding indicates that the KOR isrequired for the full effect of nalfurafine at promoting full recoverybut not disease reduction.

Example 25: Myelination is Improved in Mice Treated with Nalfurafineafter the Onset of Paralysis in the EAE Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIGS. 25A-D. On the day of diseaseonset (score ≥1), mice were started on daily treatment with vehicle onlyor nalfurafine 0.01 mg/kg by i.p. injection. During the chronic phase(>24 days post treatment initiation), mice were culled, perfused with 4%paraformaldehyde and spinal cords were processed for histology. Sectionswere stained with luxol fast blue to assess demyelination. The region ofinterest taken for analysis is shown in 25A. Note the presence ofdemyelinated regions (lesions) with less luxol fast blue (LFB) staining(myelin) in the ventral horn in EAE mice receiving vehicle (circle—25B)and no demyelinated lesions present in mice treated with nalfurafine(25C). Quantified data is shown in 25D. For each image, 5 randomisedregions of the ventral horn of the spinal cord were analysed in ImageJusing mean grey value and integrated pixel density as an indicator ofmyelin density. Data is from two individual experiments with n=4(vehicle), n=8 (nalfurafine) EAE mice respectively. Scale bar=50 μm.*p<0.05 by students t-test.

Interpretation and impact: EAE disease induces extensive lesions in thespinal cord (see vehicle only (25B), characterised by a loss of myelinand neurodegeneration, demonstrating that EAE is a destructive diseasein the CNS. Treatment of this disease state with nalfurafine reducesthis lesion load and demyelination, suggesting that treatment restoresthe spinal cord tissue to a near normal state by remyelination.

Example 26: Nalfurafine Treatment Decreases Cellular Infiltration intothe Spinal Cord when Administered Therapeutically in the EAE Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIGS. 26A-C. On the day of diseaseonset (score >1), mice were started on daily treatment with vehicle onlyor nalfurafine 0.01 mg/kg by i.p. injection. During the chronic phase(>24 days post treatment initiation), mice were culled, perfused with 4%paraformaldehyde and spinal cords were paraffin embedded for histology.10 μM coronal sections were stained with Hematoxylin and Eosin (H&E) toassess of leucocyte infiltration, a marker of inflammation withinlesions induced in EAE disease. Note the large number of leucocytespresent in the ventral horn of vehicle treated EAE mice, than in EAEmice administered nalfurafine. Images were scored by a blinded observerfor the level of infiltration on a scale ranging from 0 (noinfiltration) to 3 (maximum infiltration). Data is from two individualexperiments: n=7 mice (11 sections) for EAE Vehicle; and n=9 mice (13sections) for EAE mice treated with nalfurafine. Scale bar=50 μm).Students t-test, *p<0.05.

Interpretation and impact: EAE disease induces substantialhistopathology in the spinal cord. H&E staining of leucocytes is anindicator of lesion severity, with the higher number of infiltratingcells, the more severe the lesion, including demyelination, as shown inthe vehicle only panel and by quantification. Treatment with nalfurafineshows a surprising reduction of infiltrating leucocytes, with a nearabsence of lesions and demyelination indicating that treatment mayresolve lesions and/or cause remyelination.

Example 27: Nalfurafine Treatment Reduces the Level of ActivatedAstrocytes in the Spinal Cord when Administered Therapeutically in theEAE Model of MS

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIGS. 27A-1, and 27A-2 day 17, micewere started on daily treatment with vehicle only or nalfurafine at 0.01mg/kg by i.p. injection. On day 45 after immunization to induce EAE,mice were culled, and spinal cords were processed forimmunohistochemistry (IHC). Shown in FIGS. 27A-1 and 27A-2 arerepresentative glial fibiliary acid protein (GFAP) immunolabeled cells(black staining) from coronal sections of the ventral horn of the spinalcord taken from EAE mice. The images are from 10 μM paraffin embeddedsections, stained with Rabbit anti-GFAP at (1:1000, DAKO) before beingphotographed at 20× magnification, scale bar=50 μm. The number ofastrocytes per section in a standard ROI were counted using the cellcounter plug-in in ImageJ. Two sections were assessed per animal.Sections assessed n=7 (10-13 sections, from 2 individual experiment.***p=0.0003 (FIG. 27B).

Interpretation and impact: As shown and quantified in FIGS. 27A-1 andA-2, at day 45, there was significant elevation in the activated GFAP+astrocytes in the spinal cord of vehicle treated EAE mice. Astrocytesare recognized to be early and highly active players during lesionformation and key for providing peripheral immune cells access to thecentral nervous system (Ponath et al. The Role of Astrocytes in MultipleSclerosis. Front Immunol. 2018; 9: 217). Treatment with 0.01 mg/kg i.p.nalfurafine significantly reduces the number of activated astrocytessuggesting that nalfurafine treatment can have a neuroprotective andanti-inflammatory effect on the spinal cord tissue in the disease state(FIG. 27B).

Example 28: Nalfurafine Treatment Enhances Recovery from Weight Losswhen Administered Therapeutically in the Cuprizone Demyelination DiseaseModel of MS

FIG. 28A shows a time course of cuprizone induced demyelination andtreatment regime.

Experimental details: A demyelinating disease state was induced infemale C57BL/6 mice (8-14 weeks old and between 17-23 grams in weight).As shown in the timeline of FIG. 28A, the mice were fedcuprizone-containing chow (0.3% (w/w) cuprizone) or chow only (normalcontrols) for 35 days, at which point they were switched back tostandard chow. At day 28, mice were started on daily treatment withvehicle only (DMSO: Tween 80: Saline) or nalfurafine at 0.1 mg/kg byi.p. injection or U-50488 at 1.6 mg/kg by i.p. injection. On day 70,mice were culled and brain tissue were processed for transmissionelectron microscopy (TEM). Mice were weighed daily and the % weightchange calculated.

Interpretation and impact: This model is well established as a tool forthe study of non-immune system induced demyelination. This model enablesthe assessment of putative remyelination-promoting therapeutics(Matsushima and Morell, 2001. The neurotoxicant, cuprizone, as a modelto study demyelination and remyelination in the central nervous system.Brain Pathol. 11, 107-116).

FIG. 28B shows cuprizone induced weight change over the time course ofstudy.

Experimental details: A demyelinating disease state was induced infemale C57BL/6 mice as described in Example 28 and illustrated in FIGS.28A-C.

Interpretation and impact: Mice treated with 0.3% cuprizone (CPZ) loseweight as the disease is induced, compared to mice with normal diet,corresponding to disease induction and severity.

FIG. 28C shows that nalfurafine treatment enhances weight gain in therecovery phase of the cuprizone demyelination disease model of MS,whereas U-50488 does not.

Experimental details: A demyelinating disease state was induced infemale C57BL/6 mice as described in FIG. 28C. Diseased animals weretreated with Vehicle only, nalfurafine (0.1 mg/kg), U-50488 (1.6 mg/kg)as described in FIG. 28A. Mice were weighed daily and the % weightchange calculated. *p<0.05=nalfurafine treated mice; #p<0.05=U-50488treated mice. Two-way repeated measures ANOVA, followed by Turkey'smultiple comparison tests. (n=15 mice/group from 3 experimentalreplicates. ANOVA revealed a significant interaction F(40, 600)=2.212(p<0.0001) with significant time F(8, 600)=101.2 (p<0.0001) andtreatment effects F(5,75)=5.52 (P<0.0002).

Interpretation and impact: Mice treated with 0.3% cuprizone (CPZ) loseweight as the disease is induced. Mice recover when returned to normalchow (removal of cuprizone) (FIG. 28C). Treatment with nalfurafineenhances recovery of the lost weight faster compared to mice withvehicle only or treatment with U-50488.

Example 29: Nalfurafine Treatment Enhances Remyelination whenAdministered after Demyelination in the Cuprizone Demyelination DiseaseModel of MS

Experimental details: A demyelinating disease state was induced infemale C57BL/6 mice as illustrated in FIG. 28A. The results are shown inFIGS. 29A-G. Panels A-D of FIG. 29 show representative TransmissionElectron Microscopy (TEM) images of the corpus callosum of mice (A) fednormal diet and (B-D) fed 0.3% cuprizone to induce demyelination.Following the time course shown in FIG. 28A, cuprizone fed mice wereadministered (B) vehicle only treatment, (C) U-50488 (1.6 mg/kg/i.p.)and (D) nalfurafine (0.1 mg/kg/i.p.) and then sacrificed on experimentalday 70. Scale bars represent 2000 nm.

FIG. 29E shows the quantification and analysis of the g-ratios shows asignificant difference between treatment groups F(3,953)=21.18(p<0.0001). Mice fed a normal diet have a mean g-ratio of 0.78±0.09 incontrast to mice fed 0.3% cuprizone that have a significant increase ing-ratio of 0.84±0.1 corresponding to the decreased myelin thickness(####p<0.0001). Mice fed a diet with cuprizone treated with nalfurafine(0.1 mg/kg/i.p.) (0.75±0.15) show a significant reduction in g-ratiocompared to Vehicle treated controls (****p<0.0001), corresponding to anincreased myelin thickness. Mice fed a diet with cuprizone treated withU-50488 show a somewhat increased myelin thickness compared tovehicle-treated controls with a mean g-ratio of (0.80) (**p<0.01), but,surprisingly, nalfurafine treatment showed a significant increase inmyelin thickness (decrease in g-ratio) compared to mice treated withU-50488 (1.6 mg/kg/i.p.) ({circumflex over ( )}{circumflex over( )}{circumflex over ( )}p<0.001), indicating that nalfurafine issignificantly more effective at increasing myelin thickness thanU-50488. Data represents measurements of 5 TEM images of the corpuscallosum from two-three mice per treatment group and g-ratios calculated(a measure of myelin thickness) using Image J software. Analysis wasperformed by individuals blinded to treatment groups. (n=204-267 axonsper treatment group).

FIG. 29F shows the quantification and analysis of the number ofmyelinated axons vs non-myelinated axons in a region of interest (390μm²). n=20 images per treatment group (from n=2-3 mice).

FIG. 29G shows the quantification and analysis of the area of myelinstaining per TEM image was performed using Image 3 software (20 imagesper treatment from n=2-3 mice sacrificed on day 70). TEM images werecolour inverted (myelin white) and a threshold used to reveal myelin.The area of this myelin threshold measured for each treatment group.

All data analysed by one-way ANOVA followed by Turkeys multiplecomparisons test. Significant differences compared to vehicle only aredepicted by *; between normal mice and cuprizone/vehicle treated mice #;and between nalfurafine and U-50488 by {circumflex over ( )}. (*p<0.05;**p<0.01; ***p<0.001; ****P<0.0001).

Interpretation and impact: As shown in FIGS. 29A-G, demyelination wasvery apparent in the corpus collosum of the brain of cuprizone-induced,vehicle only treated animals (Panel B). The ratio between axonalcircumference and myelin circumference (g-ratio) decreases with normalmyelination. The cuprizone induced animals treated with nalfurafine showa more normal axonal-myelin structure, the myelinated axons are denselypacked within white matter and the myelin sheaths of neighboring fibersoften directly touch. The staining of the myelin sheaths (black) is moreprominent indicating increased remyelination. Ultrastructurally, thisnalfurafine tissue is surprisingly similar to that of the naïve (normal)tissue. Quantitatively, the nalfurafine tissue has a significantly lowerg-ratio compared to vehicle only treated indicative of enhancedremyelination, with a g-ratio closer to that of naïve (normal) tissue.This is further supported by analysis of the percentage increase in thenumber of myelinated axons and percentage increase in area ofmyelination in the nalfurafine treated animals. In contrast, treatmentwith the compound U-50488 did not show repair or restoration to a nearnormal state. Qualitatively, the axonal-myelin structure isdisorganised, there is a loss of axons, and overtly enlargedaxonal-myelin structures. Quantitatively, U-50488 treatment has outcomessimilar to that of the vehicle only treated samples (i.e. no discernibleremyelination), whereas nalfurafine treatment shows similar outcomes tothe naïve tissue. Qualitatively and quantitatively, nalfurafinetreatment enhances remyelination that is indicative of a near-fullrecovery following a demyelination insult of cuprizone.

Example 30: Nalfurafine is More Effective at Promoting FunctionalRecovery than Clemastine Fumarate, a Known Remyelinating Drug

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIGS. 30A-B. On the day of diseaseonset (score >1, dotted line), mice were treated with vehicle only(daily, n=9)) or nalfurafine (0.01 mg/kg by i.p. injection daily; n=8).In a separate similar experiment, mice were treated with vehicle only(n=5) or clemastine fumarate (10 mg/kg by i.p. injection; n=7). Shownare the aligned scores from mice starting from onset/treatmentinitiation. ****p<0.0001 by two-way ANOVA NalF or clemastine compared tovehicle.

Interpretation and impact: Clemastine fumarate, an anti-histamine whichalso antagonizes the muscarinic receptor, has been shown to reducechronic disability in the EAE model when used at 10 mg/kg starting atthe time of immunization. Additionally, it has been shown to enhanceremyelination in mice and humans (Li et al. 2015, Clemastine rescuesbehavioral changes and enhances remyelination in the cuprizone mousemodel of demyelination. Neurosci Bull.; 31: 617-625; Green, A. J et al.,2017 Clemastine fumarate as a remyelinating therapy for multiplesclerosis (ReBUILD): a randomised, controlled, double-blind, crossovertrial. Lancet Lond. Engl. 390, 2481-2489). In our EAE model, clemastineis similarly effective to previously published reports, but is much lesseffective than nalfurafine at enabling full functional recovery (Mei, F.et al. 2016, Accelerated remyelination during inflammatory demyelinationprevents axonal loss and improves functional recovery. ELife 5). Thisexample shows that nalfurafine is superior to clemastine fumarate inthis model.

Example 31: Nalfurafine Promotes a Greater and More Sustained Recoverythan Clemastine Fumarate, a Known Remyelinating Drug

Experimental detail: EAE was induced in female C57BL/6 mice as describedin Example 1. Results are shown in FIGS. 31A-1, 31A-2, 31B-1 and 31B-2.On the day of disease onset (score >1, dotted line), mice were treatedwith vehicle only (daily, n=9)) or nalfurafine (0.01 mg/kg by i.p.injection daily; n=8)(A). In a separate similar experiment, mice weretreated with vehicle only (n=5) or clemastine fumarate (10 mg/kg by i.p.injection; n=7) (B). Mice were considered recovered if they received ascore <0.5 by day 23 post treatment initiation. Shown are the percentageof mice in each group that recovered (A) and the number of days inrecovery (B). ****p<0.0001 by Fisher's exact test (% recovered; A) orone-way ANOVA with Holm-Sidak's multiple comparison test (#days inrecovery; B). These data are from the same experiments as Example 30.

Interpretation and impact: Clemastine fumarate, an anti-histamine whichalso antagonizes the muscarinic receptor, has been shown to reducechronic disability in the EAE model when used at 10 mg/kg starting atthe time of immunization. Additionally, it has been shown to enhanceremyelination in mice and humans. In our EAE model, clemastine fumaratetreatment promotes recovery in just over 50% of the mice but therecovery is not sustained. In contrast, all of the mice recover whentreated with nalfurafine and have a sustained recovery. This findingindicates that nalfurafine is superior to clemastine fumarate in thismodel and provides a more sustained improvement in every animal treated.

Example 32: Nalfurafine Promotes Recovery in Pain Threshold whenAdministered after Demyelination in the Cuprizone Demyelination DiseaseModel of MS

Experimental detail: A demyelinating disease state was induced in femaleC57BL/6 mice (8-14 weeks older and between 17-23 grams in weight). Themice were fed cuprizone-containing chow (0.3% (w/w) cuprizone) or chowonly (normal controls) for 35 days, at which point they were switchedback to standard chow. At day 28, mice were started on daily treatmentwith vehicle only (DMSO: Tween 80: Saline) or nalfurafine at 0.1 mg/kgby i.p. injection. In a second experiment, mice were fedcuprizone-containing chow (0.3% (w/w) cuprizone) or chow only (normalcontrols) for 42 days, at which point they were switched back tostandard chow. At day 35, mice were started on daily treatment withvehicle only (DMSO: Tween 80: Saline) or nalfurafine at 0.1 mg/kg byi.p. injection. In both studies, on day 70, mice were culled. See FIG.32 A for an outline of the disease induction and treatment time course.

Sensitivity to mechanical force elicits paw withdrawal in mice.Threshold to withdrawal is measured using calibrated von Frey filamentsusing the up-down method (Bonin et al. A simplified up-down method(SUDO) for measuring mechanical nociception in rodents using von Freyfilaments. Molecular Pain. 2014; 10:1-11) at maximum disease, prior totreatment with nalfurafine. Cuprizone causes increased mechanicalsensitivity compared to mice on a normal diet ({circumflex over( )}p<0.05) (FIG. 32B), and this increase in mechanical withdrawalthreshold is reduced to baseline levels following treatment withnalfurafine (0.1 mg/kg/i.p.). *p<0.05 at maximum disease (day 28 or 35)and following daily treatment with nalfurafine (average threshold days45-70). Nalfurafine treated mice improved mechanical threshold scorescompared to vehicle treated mice (#p<0.05). Student t-test, n=10-11mice/group from 2 independent experiments. {circumflex over ( )}comparedto mice on normal diet; * threshold pre and post treatment; #differencesin recovery between treatment groups. Pooled data from 2 experimentalcohorts were analysed (max disease is week prior to treatment initiationand at maximal recovery (days 63-70).

Interpretation and impact: Chronic pain is often associated withmultiple sclerosis. Allodynia is an increase in pain sensation to anormally non-painful stimulus. In this test von Frey filaments are usedto measure the paw withdrawal threshold following application of adefined mechanical force. Following cuprizone induced demyelination, thepain threshold is a functional biomarker for recovery, indicative ofremyelination of the nerve fibres. Remarkably, the diseased animalstreated with nalfurafine showed a pain sensitivity that was similar tobaseline, indicating that treatment enhances functional recovery.

The claims defining the invention are as follows:
 1. A method oftreating multiple sclerosis (MS) in a human subject in need thereof, themethod comprising identifying a human subject as having MS; andadministering to the human subject daily, for a period of at least 7days, a pharmaceutical composition comprising a therapeuticallyeffective amount of about 2.5 μg to about 83.0 μg of nalfurafine,wherein the therapeutically effective amount is sufficient to increaseremyelination in the human subject, wherein the increase inremyelination comprises increasing a percentage of myelination area inthe human subject's brain to a level that is up to about 89% of apercentage of myelination area in a healthy human brain.
 2. The methodof claim 1, wherein the daily dose of nalfurafine is 10.0 μgnalfurafine.
 3. The method of claim 1, wherein the method comprisesdaily administration of the pharmaceutical composition for at least 14days.
 4. The method of claim 1, wherein the method comprisesadministration of the pharmaceutical composition for at least one month.5. The method of claim 1, further comprising one or more of thefollowing steps selected from the group consisting of: diagnosing MS inthe subject; testing for remyelination in the subject; testing for alevel of paralysis in the subject; testing coordination in the subject;and testing balance in the subject.
 6. A method of increasingremyelination in a human subject having multiple sclerosis (MS), themethod comprising identifying a subject as having MS; and administeringto the subject for a period of at least 7 days a pharmaceuticalcomposition comprising an amount of nalfurafine effective to increase alevel of remyelination in the subject relative to a level of myelinationin the subject before administering the pharmaceutical composition,wherein the amount of nalfurafine in the pharmaceutical compositioncomprises about 0.1 to about 10.0 μg nalfurafine.
 7. The method of claim6, wherein the method results in one or more clinical outcomes selectedfrom the group consisting of: a decrease in MS disease progression; adecrease in MS disease severity; a decrease or delay in nerve celldemyelination; a decrease in frequency or severity of relapsing MSattacks; a healing of damaged nerve tissue; an increase in remyelinationof demyelinated nerves in the subject's central nervous system;protection of damaged nerve tissue from further disease activity;promotion of neuronal outgrowth in the subject's central nervous system;an improvement in nerve function; and an enhanced rate of remission. 8.The method claim 6, wherein the method results in a reduction of one ormore clinical symptoms of MS selected from the group consisting of: lossof sensitivity, muscle weakness, impaired walking, impaired handfunction, pronounced reflexes, muscle spasms, difficulty in moving,ataxia, spasticity, problems with speech or swallowing, visual problems,fatigue, acute or chronic pain, facial pain, incontinence, reducedcognitive ability, depression, anxiety, sexual dysfunction, Uhthoff'sphenomenon, and Lhermitte's sign.
 9. The method of claim 6, wherein themethod comprises daily administration of the pharmaceutical compositionfor at least 14 days.
 10. The method of claim 6, wherein the methodcomprises administration of the pharmaceutical composition for at leastone month.
 11. The method of claim 6, further comprising one or more ofthe following steps selected from the group consisting of: diagnosing MSin the subject; testing for demyelination in the subject; testing for areduction in demyelination in the subject; testing for remyelination inthe subject; testing for a level of paralysis in the subject; testingcoordination in the subject; and testing balance in the subject.
 12. Amethod of attenuating demyelination in a subject having multiplesclerosis (MS), the method comprising identifying a subject as havingMS; and administering to the subject for a period of at least 7 days apharmaceutical composition comprising an amount of nalfurafine effectiveto decrease a level of demyelination relative to a level ofdemyelination before administering the pharmaceutical composition,wherein the amount of nalfurafine in the pharmaceutical compositioncomprises about 0.1 to about 10.0 μg nalfurafine.
 13. The method ofclaim 12, wherein the method results in one or more clinical outcomesselected from the group consisting of: a decrease in MS diseaseprogression; a decrease in MS disease severity; a decrease or delay innerve cell demyelination; a decrease in frequency or severity ofrelapsing MS attacks; a healing of damaged nerve tissue; an increase inremyelination of demyelinated nerves in the subject's central nervoussystem; protection of damaged nerve tissue from further diseaseactivity; promotion of neuronal outgrowth in the subject's centralnervous system; an improvement in nerve function; and an enhanced rateof remission.
 14. The method claim 12, wherein the method results in areduction of one or more clinical symptoms of MS selected from the groupconsisting of: loss of sensitivity, muscle weakness, impaired walking,impaired hand function, pronounced reflexes, muscle spasms, difficultyin moving, ataxia, spasticity, problems with speech or swallowing,visual problems, fatigue, acute or chronic pain, facial pain,incontinence, reduced cognitive ability, depression, anxiety, sexualdysfunction, Uhthoff's phenomenon, and Lhermitte's sign.
 15. The methodof claim 12, wherein the method comprises daily administration of thepharmaceutical composition for at least 14 days.
 16. A method ofreducing one or more clinical symptoms of multiple sclerosis (MS) in ahuman subject having MS, the method comprising identifying a subject ashaving MS; and administering to the subject for a period of at least 7days a pharmaceutical composition comprising an amount of nalfurafineeffective to decrease a clinical symptom in the subject relative to alevel of the clinical symptom in the subject before administering thepharmaceutical composition, wherein the amount of nalfurafine in thepharmaceutical composition comprises about 0.1 to about 10.0 μgnalfurafine.
 17. The method of claim 16, wherein the method furtherresults in one or more clinical outcomes selected from the groupconsisting of: a decrease in MS disease progression; a decrease in MSdisease severity; a decrease or delay in nerve cell demyelination; adecrease in frequency or severity of relapsing MS attacks; a healing ofdamaged nerve tissue; an increase in remyelination of demyelinatednerves in the subject's central nervous system; protection of damagednerve tissue from further disease activity; promotion of neuronaloutgrowth in the subject's central nervous system; an improvement innerve function; and an enhanced rate of remission.
 18. The method claim16, wherein the method results in a reduction of one or more clinicalsymptoms of MS selected from the group consisting of: loss ofsensitivity, muscle weakness, impaired walking, impaired hand function,pronounced reflexes, muscle spasms, difficulty in moving, ataxia,spasticity, problems with speech or swallowing, visual problems,fatigue, acute or chronic pain, facial pain, incontinence, reducedcognitive ability, depression, anxiety, sexual dysfunction, Uhthoff'sphenomenon, and Lhermitte's sign.
 19. The method of claim 16, whereinthe method comprises daily administration of the pharmaceuticalcomposition for at least 14 days.
 20. The method of claim 1, wherein thedaily dose of nalfurafine is about 8.3 μg to 83.0 μg of nalfurafine. 21.The method of claim 1, wherein the daily dose of nalfurafine is about2.5 μg to 25 μg.